Combination Therapy for Treating Cancer With an Antibody and Intravenous Administration of a Recombinant MVA

ABSTRACT

The invention relates to a pharmaceutical combination and related methods for reducing tumor volume and/or increasing the survival of a cancer patient. The combination comprises an intravenous administration of a recombinant MVA encoding a tumor-associated antigen and an administration of an antibody to a cancer patient.

FIELD OF THE INVENTION

The present invention relates to a combination therapy for the treatmentof cancers, the combination including an intravenously administeredrecombinant modified vaccinia Ankara (MVA) virus comprising a nucleicacid encoding a heterologous tumor-associated antigen (TAA) and anantibody.

BACKGROUND OF THE INVENTION

Recombinant poxviruses have been used as immunotherapy vaccines againstinfectious organisms and, more recently, against tumors (see Mastrangeloet al. (2000) J Clin Invest. 105(8): 1031-1034).

One poxviral strain that has proven useful as an immunotherapy vaccineagainst infectious disease and cancer is the Modified Vaccinia Ankara(MVA) virus. MVA was generated by 516 serial passages on chicken embryofibroblasts of the Ankara strain of vaccinia virus (CVA) (for review seeMayr et al. (1975) Infection 3: 6-14). As a consequence of theselong-term passages, the genome of the resulting MVA virus had about 31kilobases of its genomic sequence deleted and, therefore, was describedas highly host cell restricted for replication to avian cells (Meyer etal. (1991) J. Gen. Virol. 72: 1031-1038). It was shown in a variety ofanimal models that the resulting MVA was significantly avirulent (Mayr &Danner (1978) Dev. Biol. Stand. 41: 225-34). Strains of MVA havingenhanced safety profiles for the development of safer products, such asvaccines or pharmaceuticals, have been described (See International PCTpublication WO2002042480; see also, e.g., U.S. Pat. Nos. 6,761,893 and6,913,752, all of which are incorporated by reference herein). Suchvariants are capable of reproductive replication in non-human cells andcell lines, especially in chicken embryo fibroblasts (CEF), but arereplication incompetent in human cell lines, in particular includingHeLa, HaCat and 143B cell lines. Such strains are also not capable ofreproductive replication in vivo, for example, in certain mouse strains,such as the transgenic mouse model AGR 129, which is severelyimmune-compromised and highly susceptible to a replicating virus (seeU.S. Pat. No. 6,761,893). Such MVA variants and its derivatives,including recombinants, referred to as “MVA-BN,” have been described(see International PCT publication WO2002042480; see also, e.g., U.S.Pat. Nos. 6,761,893 and 6,913,752).

The use of poxviral vectors that encode tumor-associated antigens (TAAs)have been shown to successfully reduce tumor size as well as increaseoverall survival rate of cancer patients (see, e.g., WO 2014/062778). Ithas been demonstrated that when a cancer patient is administered apoxviral vector encoding a TAA, such as HER2, CEA, MUC1, and/orBrachyury, a robust and specific T-cell response is generated by thepatient to fight the cancer (Id; see also, Guardino et al. (2009) CancerRes. 69: Abstract 5089, Heery et al. (2015) JAMA Oncol. 1: 1087-95).

HER2 is one such TAA that has been shown to be effective when encoded aspart of a poxviral vector (Id.). HER2 is a tumor-associated antigen thatis over-expressed in certain types of tumor cells in some patientshaving different types of cancer, such as breast, colorectal, lung,ovarian, cervical, bladder, gastric, and urothelial cancers.Immunization with various HER2 polypeptides has been used to generate animmune response against tumor cells expressing this antigen, as hasvaccination with recombinant modified vaccinia virus Ankara (“MVA”)vectors expressing a modified form of the HER2 protein (i.e.,MVA-BN-HER2). (See, e.g., Renard et al. (2003) J. Immunol.171:1588-1595; Mittendorf et al. (2006) Cancer 106: 2309-2317; Mandl etal. (2012) Cancer Immunol. Immunother. 61(1):19-29).

Previous work with MVA-BN-HER2 showed that it induced a THi1-biasedimmune response having both antibody and cellular components (see, e.g.,Mandl et al. (2012)). Some workers have shown that a balanced immuneresponse including both humoral and cell-mediated components isimportant for protection from and clearance of a variety of pathogens inthe context of infectious disease (see, e.g., Hutchings et al. (2005) J.Immunol. 175: 599-606).

In addition to their effectiveness with TAAs, poxviruses, such as MVAhave been shown to have enhanced efficacy when combined with a CD40agonist such as CD40 Ligand (CD40L) (see WO 2014/037124). CD40/CD40L isa member of the tumor necrosis factor receptor/tumor necrosis factor(“TNFR/TNF”) superfamily. While CD40 is constitutively expressed on manycell types, including B-cells, macrophages and DCs, its ligand CD40L ispredominantly expressed on activated CD4+ T-cells (Lee et al. (2002) JImmunol. 171(11): 5707-5717; Ma and Clark (2009) Semin. Immunol.21(5):265-272). The cognate interaction between DCs and CD4+ T-cellsearly after infection or immunization ‘licenses’ DCs to prime CD8+T-cell responses (Ridge et al. (1998) Nature 393(6684): 474-478). DClicensing results in the upregulation of co-stimulatory molecules,increased survival and better cross-presenting capabilities of DCs. Thisprocess is mainly mediated via CD40/CD40L interaction (Bennet et al.(1998) Nature 393(6684): 478-480; Schoenberger et al. (1998) Nature393(6684): 480-483), but CD40/CD40L-independent mechanisms also exist(CD70, LT.beta.R). Interestingly, a direct interaction between CD40Lexpressed on DCs and CD40 expressed on CD8+ T-cells has also beensuggested, providing a possible explanation for the generation ofhelper-independent CTL responses (Johnson et al. (2009) Immunity 30(2):218-227).

Several studies indicate that agonistic anti-CD40 antibodies may beuseful as a vaccine adjuvant. In addition, recombinant AdV (Kato et al.(1998) J. Clin. Invest. 101(5): 1133-1141) and VV (Bereta et al. (2004)Cancer Gen. Ther. 11(12): 808-818) encoding CD40L have been created thatshowed superior immunogenicity in vitro and in vivo compared tonon-adjuvanted viruses.

CD40L, when encoded as part of an MVA, was shown to be able to induceand enhance the overall T-cell response for a disease associated antigen(WO 2014/037124). In WO 2014/037124 it was shown that a recombinant MVAencoding CD40L and a heterologous antigen was able to enhance DCactivation in vivo, increase T-cell responses specific to theheterologous antigen and enhance the quality and quantity of CD8 T-cells(Id.).

The use of antibodies for cancer therapy has also seen considerablesuccess in the past decade (see, e.g., Scott et al. (2012) NatureReviews Cancer 12: 278-287). There are several antibody therapies thathave received FDA approval and kill tumor cells in a variety of ways.For example, antibody therapies can kill tumor cells through directaction of the antibody, such as an antibody binding to a tumor antigenon the cell surface (Id.; see also Brodowicz et al. (2001) Br. J. Cancer85: 1764-70). This can lead to apoptosis and death of the tumor cell aswell as inhibition of tumor receptor activity. Preventing receptoractivity can include: preventing dimerization of the tumor receptor,preventing kinase activation, blocking of extracellular receptorcleavage, induction of receptor internalization and down-streamsignaling. The inhibition of tumor receptor activity by antibodytherapies can prevent tumor proliferation. Id.

Antibody therapies can additionally kill tumor cells, by improving acancer patient's own immune system to attack the tumor cell, termedimmune-mediated tumor cell killing (Id). Immune-mediated tumor cellkilling can include phagocytosis, complement activation,antibody-dependent cellular cytotoxicity (ADCC), genetically modifiedT-cells being targeted to the tumor by antibody, and inhibition of Tcell inhibitor receptors, such as CTLA-4 (Id).

ADCC is one of the most important ways by which antibody therapiesattack and destroy tumor cells. ADCC is triggered through interactionbetween a target-bound antibody on a tumor cell membrane and effectorcells from a patient's immune system (Wang et al. (2015) Front. Immunol.6: 368). The anti-tumor efficacy of many antibody therapies has beenshown to be Natural Killer (NK) cell dependent (Id). Human NK cells canexpress proteins that bind to the Fc portion of the antibodies. Oncebound and activated, the NK cells mediate tumor killing through severalpathways, including exocytosis of cytotoxic granules, TNF family deathreceptor signaling, and pro-inflammatory cytokine release, such as IFNγ(Id).

While there are successful poxviral cancer treatments, chemo- andradiotherapies, and antibody therapies available to cancer patients,there are many mechanisms that tumor cells employ to escape and/ordiminish these treatments. For example, in order to escape a patient'sspecific immune system, many tumor cells utilize immune checkpointmolecules and/or lower specific Major Histocompatibility Complex (MHC)expression so as to suppress and/or evade detection by the specific CD8T cells of the immune system (Scott et al. (2012)). Tumor cells haveadditionally been shown to evade a patient's innate immune response bymodifying or decreasing tumor antigen expression on the tumor cellsurface which can decrease both antibody binding, and tumor killing bythe NK cells (Id).

More recently it has been discovered that tumor cells can evade theimmune system and cancer therapies by entering an equilibrium phase witha cancer patient's immune system (see Bhatia et al. (2011) CancerMicroenvironment 4: 209-217). In at least one aspect of the equilibriumphase, tumor cells can remain in the body below the threshold ofconventional morphologic recognition or cytogenetic recognition (Id).For example, expression of tumor antigen receptors on tumor cells willfluctuate and, in many cases, decrease to a point that is below thethreshold at which the immune system can recognize the tumor cell (Id).

Given the ability of cancers and tumor cells to actively evade cancertherapies and a patient's immune system, there exists a substantial needfor developing cancer treatments that effectively target and kill thosetumor cells that actively evade the immune system. Additionally, thereexists a need for cancer treatments that can attack and kill tumors andtumor cells that utilize an equilibrium phase to evade therapies andimmune systems. At least in one aspect, the various embodiments of thepresent invention successfully overcome difficulties involving treatingtumor cells that actively evade the immune system.

BRIEF SUMMARY OF THE INVENTION

It was determined in the various embodiments of the present inventionthat a recombinant MVA when administered intravenously to a patient incombination with antibody against a tumor surface antigen enhancestreatment of the cancer patient, more particularly increases reductionin tumor volume and/or increases survival of the cancer patient.

Accordingly, in one embodiment, the present invention includes apharmaceutical combination for use in reducing tumor size and/orincreasing survival in a cancer patient, the pharmaceutical combinationcomprising: a) a recombinant modified vaccinia virus Ankara (MVA)comprising a first nucleic acid encoding a first heterologoustumor-associated antigen (TAA) that when administered intravenouslyinduces both an enhanced Natural Killer (NK) cell response and anenhanced T cell response in the cancer patient as compared to a NK celland T cell response induced by a non-intravenous administration of arecombinant MVA comprising a nucleic acid encoding a heterologoustumor-associated antigen; and b) an antibody, wherein the antibodycomprises an Fc domain and is specific to an antigen that is expressedon the cell membrane of a tumor cell; wherein administration of a) andb) to the cancer patient reduces tumor size and/or increases thesurvival rate of the cancer patient as compared to a non-intravenousadministration of either a) or b) alone. In additional embodiments, therecombinant MVA further comprises a second nucleic acid encoding asecond heterologous TAA.

In one or more preferred embodiments, the pharmaceutical combinationfurther comprises CD40L. In a most preferred embodiment, the CD40L isencoded by the recombinant MVA.

In various embodiments, the antibody is approved for the treatment of acancer patient. In one or more particular embodiments, the antibody isselected from the group consisting of: anti-CD20 (e.g., rituximab,ofatumumab, tositumomab); Anti-CD52 (e.g., alemtuzumab, Campath®antibody); anti-EGFR (e.g., cetuximab (Erbitux® antibody), panitumumab);anti-CD2 (e.g., siplizumab); anti-CD37 (e.g., BI836826); anti-CD123(e.g., JNJ-56022473); anti-CD30 (e.g., XmAb2513); anti-CD38 (e.g.,daratumumab (Darzalex® antibody)); anti-PDL1 (e.g., avelumab,atezolilzumab, durvalumab); anti-CTLA-4 (e.g., ipilumumab); anti-GD2(e.g., 3F8, ch14.18, KW-2871, dinutuximab); anti-CEA; anti-MUC1;anti-FLT3; anti-CD19; anti-CD40; anti-SLAMF7; anti-CCR4; anti-B7-H3;anti-ICAM1; anti-CSF1R; anti-CA125 (e.g., oregovomab), anti-FRα (e.g.,MOv18-IgG1, mirvetuximab soravtansine (IMGN853), MORAb-202);anti-mesothelin (e.g., MORAb-009); and anti-HER2. In a more preferredembodiment, the antibody is an anti-HER2 antibody. In a most preferredembodiment, the antibody is an anti-HER2 antibody selected frompertuzumab, trastuzumab, Herzuma® antibody, ABP 980, and ado-trastuzumabemtansine.

In various additional embodiments, the first and/or second TAA comprisesone or more mutations to prevent the first and/or second TAA frombinding and/or interacting with the antibody of the combination therapy.In one or more preferred embodiments, the first TAA is a HER2 antigen.In a more preferred embodiment the HER2 antigen comprises one or moremutations to prevent the binding of the first TAA to the anti-HER2antibody. In additional preferred embodiments, the second TAA is aBrachyury antigen. In a more preferred embodiment, the Brachyury antigencomprises one or more mutations to the nuclear localization signaling(NLS) domain.

In one or more preferred embodiments, the recombinant MVA is MVA-BN or aderivative thereof.

In various embodiments, the present invention is directed to one or moremethods of reducing tumor size and/or increasing survival of a cancerpatient. In one embodiment, there is a method comprising: (a)intravenously administering to the cancer patient a recombinant MVAcomprising a first nucleic acid encoding a first heterologoustumor-associated antigen (TAA) that when administered intravenouslyinduces both an enhanced Natural Killer (NK) cell response and anenhanced T cell response in the cancer patient as compared to an NK cellresponse and a T cell response induced by a non-intravenousadministration of a recombinant MVA virus comprising a nucleic acidencoding a heterologous tumor-associated antigen; and (b) administeringto the cancer patient an antibody, wherein the antibody comprises an Fcdomain and is specific to an antigen that is expressed on the cellmembrane of a tumor cell; wherein administration of (a) and (b) to thecancer patient reduces tumor size in the cancer patient and/or increasesthe survival rate of the cancer patient as compared to a non-intravenousadministration of either (a) or (b) alone.

In one or more preferred embodiments, the method comprises intravenouslyadministering CD40L to the cancer patient. In a more preferredembodiment the CD40L is encoded by the recombinant MVA.

In another embodiment, the recombinant MVA of present invention isadministered at the same time or after administration of the antibody.In a more preferred embodiment, the recombinant MVA is administeredafter the antibody.

In yet another embodiment, the present invention includes a method forenhancing antibody therapy in a cancer patient, the method comprisingadministering the pharmaceutical combination of the present invention toa cancer patient, wherein administering the pharmaceutical combinationenhances antibody dependent cell-mediated cytotoxicity (ADCC) induced bythe antibody therapy, as compared to administering the antibody therapyalone.

In still another embodiment, there is a method for inducing both anenhanced innate and an enhanced adaptive immune response in a cancerpatient comprising administering a pharmaceutical combination of thepresent disclosure, wherein administering the pharmaceutical combinationenhances both the innate and adaptive immune responses of the cancerpatient as compared to a non-intravenous administration of thepharmaceutical combination or elements of the combination by themselves.

In still various additional embodiments, the present invention isdirected to one or more synthetic peptides and nucleic acids encodingthe synthetic peptides. In more specific embodiments, there is asynthetic HER2 peptide and nucleic acid. In more preferred embodiments,the synthetic HER2 peptide includes one or more mutations that preventthe HER2 peptide from binding a HER2 antibody, preferably an antibodyselected from pertuzumab, trastuzumab, Herzuma® antibody, ABP 980, andado-trastuzumab emtansine. In additional preferred embodiments, thesynthetic HER2 peptide includes one or more mutations that preventextracellular dimerization, tyrosine kinase activity, and/orphosphorylation of the HER2 antigen.

In another more specific embodiment, there are one or more syntheticBrachyury peptides and nucleic acids. In more preferred embodiments, thesynthetic Brachyury polypeptides and nucleic acids include one or moremutations in the nuclear localization signaling (NLS) domain.

Additional objects and advantages of the invention will be set forth inpart in the description which follows, and in part will be obvious fromthe description or may be learned by practice of the invention. Theobjects and advantages of the invention will be realized and attained bymeans of the elements and combinations particularly pointed out in theappended claims.

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate one or more embodiments of theinvention and together with the description, serve to explain theprinciples of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1G show that intravenous (IV) administration of MVA-OVA (rMVA)leads to a stronger systemic activation of NK cells as compared tosubcutaneous (SC) administration. NK cell activation is further enhancedwhen the MVA encodes CD40L (rMVA-CD40L). Shown are the results ofExample 1, wherein staining to assess NK cell frequencies and expression(shown as Geometric Mean Fluorescence Intensity (GMFI)) of the namedprotein markers in NKp46⁺CD3⁻ cells was assessed in the spleen. FIG. 1A:NKp46′ CD3⁻ cells; FIG. 1B: CD69; FIG. 1C: NKG2D; FIG. 1D: FasL; FIG.1E: Bcl-X_(L); FIG. 1F: CD70; and FIG. 1G: IFN-γ.

FIGS. 2A-2G show that IV administration of MVA-OVA (rMVA) leads to astronger systemic activation of NK cells as compared to SCadministration. NK cell activation is further enhanced when the MVAencodes CD40L (rMVA-CD40L). Shown are the results of Example 1, whereinstaining to assess NK cell frequencies and expression (shown asGeometric Mean Fluorescence Intensity (GMFI)) of the named proteinmarkers in NKp46+CD3⁻ cells was assessed in the liver. FIG. 2A: NKp46⁺CD3⁻ cells; FIG. 2B: CD69; FIG. 2C: NKG2D; FIG. 2D: FasL; FIG. 2E:Bcl-X_(L); FIG. 2F: CD70; and FIG. 2G: IFN-γ.

FIGS. 3A-3G show that IV administration of MVA-OVA (rMVA) leads to astronger systemic activation of NK cells as compared to SCadministration. NK cell activation is further enhanced when the MVAencodes CD40L (rMVA-CD40L). Shown are the results of Example 1, whereinstaining to assess NK cell frequencies and expression (shown asGeometric Mean Fluorescence Intensity (GMFI)) of the named proteinmarkers in NKp46⁺CD3⁻ cells was assessed in the lung. FIG. 3A: NKp46⁺CD3⁻ cells; FIG. 3B: CD69; FIG. 3C: NKG2D; FIG. 3D: FasL; FIG. 3E:Bcl-X_(L); FIG. 3F: CD70; and FIG. 3G: IFN-γ.

FIGS. 4A-4F show that intravenous (IV) administration ofMVA-HER2-Twist-CD40L leads to a stronger systemic activation of NK cellsas compared to subcutaneous (SC) administration. Shown are the resultsof Example 1, wherein staining to assess NK cell frequencies andexpression (shown as Geometric Mean Fluorescence Intensity (GMFI)) ofthe named protein markers in NKp46⁺CD3⁻ cells was assessed in thespleen. FIG. 4A: NKp46⁺ CD3⁻ cells; FIG. 4B: CD69; FIG. 4C: FasL; FIG.4D: Bcl-X_(L); FIG. 4E: CD70; and FIG. 4F: IFN-γ.

FIGS. 5A-5F show that IV administration of MVA-HER2-Twist-CD40L leads toa stronger systemic activation of NK cells as compared to SCadministration. Shown are the results of Example 1, wherein staining toassess NK cell frequencies and expression (shown as Geometric MeanFluorescence Intensity (GMFI)) of the named protein markers inNKp46⁺CD3⁻ cells was assessed in the liver. FIG. 5A: NKp46⁺CD3-cells;FIG. 5B: CD69; FIG. 5C: FasL; FIG. 5D: Bcl-X_(L); FIG. 5E: CD70; andFIG. 5F: IFN-γ.

FIGS. 6A-6F show that IV administration of MVA-HER2-Twist-CD40L leads toa stronger systemic activation of NK cells as compared to SCadministration. Shown are the results of Example 1, wherein staining toassess NK cell frequencies and expression (shown as Geometric MeanFluorescence Intensity (GMFI)) of the named protein markers inNKp46⁺CD3⁻ cells) was assessed in the lung. FIG. 6A: NKp46⁺ CD3-cells;FIG. 6B: CD69; FIG. 6C: FasL; FIG. 6D: Bcl-X_(L); FIG. 6E: CD70; andFIG. 6F: IFN-γ.

FIGS. 7A-7F show that IV administration of MVA-OVA-CD40L (rMVA-CD40L)leads to enhanced levels of IL-12p70 and IFN-γ. Shown are the results ofExample 2. FIG. 7A: The concentration of IFN-γ was higher afterrMVA-CD40L as compared to MVA-OVA (rMVA) immunization. FIG. 7B: The NKcell activating cytokine IL-12p70 was only detectable after MVA-CD40Limmunization. High serum levels of IFN-γ are in line with higherfrequencies of IFN-γ⁺NK cells (see FIG. 1G) and CD69⁺ granzyme B⁺ NKcells in the spleen (FIG. 7C) after rMVA-CD40L immunization. Similarresponses were seen in NHPs (Macaca fascicularis) after IV (intravenous)injection of MVA-MARV-GP-huCD40L (rMVA-CD40L), namely higher serumconcentrations of IFN-γ (FIG. 7D) and IL-12p40/70 (FIG. 7E) as well asmore proliferating (Ki67⁺) NK cells (FIG. 7F) as compared to MVA-MARV-GP(rMVA).

FIGS. 8A-8C show a time course of NK cell activation and proliferation.Shown are the results of Example 3, wherein staining to assess NK cellfrequencies and expression of the named protein markers in NKp46⁺CD3⁻cells was assessed in the spleen, liver, lung and blood. FIG. 8A:CD3⁻CD19⁻NKp46⁺; FIG. 8B: NK cell proliferation marker Ki67; and FIG.8C: CD69 expression (shown as Geometric Mean Fluorescence Intensity(GMFI)).

FIG. 9 shows enhanced NK cell mediated toxicity ex vivo upon systemicMVA-OVA (rMVA) and MVA-CD40L (rMVA-CD40L) immunization. Splenic NK cellswere purified and used as effectors in a target killing assay asdescribed in Example 4. NK cells were cultured with CFSE-labelled MHCclass I-deficient YAC-1 cells at the indicated ratios overnight.Specific killing was assessed by quantifying unviable CFSE⁺ YAC-1 cellsby flow cytometry.

FIGS. 10A-10E show enhanced antibody-dependent cellular cytotoxicity(ADCC) in vivo and ex vivo upon systemic MVA-OVA (rMVA), MVA-CD40L(rMVA-CD40L), and MVA-HER2-Twist-CD40L immunization as described inExample 5. FIG. 10A: C57BL/6 mice were treated IV either with 25 μganti-CD4, rMVA+5 μg rat IgG2b, 1 μg anti-CD4 or MVA-OVA (rMVA)+1 μganti-CD4. CD4 T cell (CD3⁺CD4⁺) depletion in the liver was analyzed andis shown as percent specific killing. To assess ex vivo ADCC activity ofNK cells, (FIG. 10B) C57BL/6 or (FIG. 10C) Balb/c mice were immunized IVeither with PBS, MVA-OVA (rMVA) or MVA-OVA-CD40L (rMVA-CD40L). FIG. 10E:Balb/c mice were immunized IV either with PBS or MVA-HER2-Twist-CD40L.Splenic NK cells were purified and used as effectors inantibody-dependent killing assays. B16.F10 cells were coated with mouseanti-human/mouse Trp1 mAb (clone TA99, FIG. 10B) and (FIG. 10C)CT26-HER2 cells were coated with mouse anti-human HER2 mAb (clone7.16.4). Purified NK cells were added to the antibody-coated targetcells at a 5:1 and 4:1 ratio, respectively. FIG. 10D: CT26-HER2 cellsincubated with different concentrations of anti-human HER2 were alsomore efficiently killed by rMVA-CD40L activated NK cells as compared torMVA activated NK cells. FIG. 10E: CT26-HER2 cells were coated withvarious concentrations of mouse anti-human HER2 mAb (clone 7.16.4).Purified NK cells were added to the antibody-coated target cells at a5:1 ratio.

FIG. 11 shows that IV immunization induces stronger CD8 T cell responsesthan SC immunization. Described in Example 6, C57BL/6 mice wereimmunized either SC or IV with MVA-OVA on days 0 and 15. OVA-specificCD8 T cell responses in the blood were assessed after staining withH-2K^(b)/OVA₂₅₇₋₂₆₄ dextramers.

FIG. 12 shows that CD8 T cell responses can be further enhanced byMVA-CD40L. Described in Example 7, C57BL/6 mice were immunized IV withMVA-OVA (rMVA) or MVA-OVA-CD40L (rMVA-CD40L) on days 0 and 35.OVA-specific CD8 T cell responses in the blood were assessed afterstaining with H-2K^(b)/OVA₂₅₇₋₂₆₄ dextramers.

FIGS. 13A-13B shows repeated NK cell activation and proliferation afterprime/boost immunization. Described in Example 8, C57BL/6 mice wereimmunized IV either with PBS, MVA-OVA (rMVA) or MVA-OVA-CD40L(rMVA-CD40L) as shown in Table 1. NK cells (NKp46⁺CD3⁻) were analyzed inthe blood by flow cytometry one and four days after second and thirdimmunization. FIG. 13A Shows GMFI CD69 and FIG. 13B shows frequency ofKi67⁺ NK cells.

FIGS. 14A-14M show systemic cytokine responses after prime/boostimmunization. Described in Example 9, C57BL/6 mice were immunized IV(intravenously) either with PBS, MVA-OVA (rMVA), or MVA-OVA-CD40L(rMVA-CD40L) as shown in Table 1. Serum cytokine levels were measured at6 hours post immunization. Shown are the results, in FIG. 14A: IL-6;FIG. 14B: CXCL10; FIG. 14C: IFN-α; FIG. 14D: IL-22; FIG. 14E: IFN-γ;FIG. 14F: CXCL1; FIG. 14G: CCL4; FIG. 14H: CCL7; FIG. 14I: CCL2; FIG.14J: CCL5; FIG. 14K: TNF-α; FIG. 14L: IL-12p70; and FIG. 14M: IL-18.

FIGS. 15A-15D show strong antigen-specific CD8 T cell responses afterMVA and MVA-CD40L prime/boost immunization. Described in Example 10,C57BL/6 mice were immunized IV either with PBS, MVA-OVA (rMVA) orMVA-OVA-CD40L (rMVA-CD40L) as shown in Table 1. Induction ofantigen-specific CD8 T cell responses after repetitive immunization wasassessed. FIG. 15A: CD8 T cell frequencies were assessed; FIG. 15B: B8specific CD8 T cell responses were assessed, FIG. 15C:Transgene-specific (OVA) responses were assessed; and FIG. 15D: Ratiosof OVA/B8-specific CD8 T cells were assessed.

FIGS. 16A-16B show CD8 and CD4 effector T cell induction after MVA andMVA-CD40L prime/boost immunization. Described in Example 11, C57BL/6mice were immunized IV either with PBS, MVA-OVA (rMVA) or MVA-OVA-CD40L(rMVA-CD40L) as shown in Table 1. Phenotypically, effector T cells wereidentified by the expression of CD44 and the lack of surface CD62L. FIG.16A: CD44⁺ CD62L⁻ CD8 T cells; and FIG. 16B: CD4 T cells in the bloodwere monitored.

FIGS. 17A-17B show superior anti-tumor effect of IV rMVA-CD40Limmunization in a heterologous prime boost scheme in a melanoma model.C57BL/6 mice bearing palpable B16.OVA tumors were primed (dotted line)either with PBS, MVA-OVA (rMVA) or MVA-OVA-CD40L (rMVA-CD40L) SC or IVas described in Example 12. Mice received subsequent boosts with FPV-OVA7 and 14 days after prime (dashed lines). Tumor growth was measured atregular intervals. Shown are tumor mean volume (FIG. 17A) and survivalof tumor-bearing mice (FIG. 17B) by day 45 after tumor inoculation.

FIG. 18 shows efficient tumor control after a single IV immunizationwith MVA-OVA-CD40L (rMVA-CD40L). C57BL/6 mice bearing palpable B16.OVAtumors were primed IV or received IV prime and boost as described inExample 13. Tumor growth was measured at regular intervals. Shown is thetumor mean volume.

FIGS. 19A-19C show that CD8 T cells are essential players in rMVA-CD40Lmediated tumor control. C57BL/6 mice bearing palpable B16.OVA tumorswere immunized IV either with PBS, MVA-OVA (rMVA) or MVA-OVA-CD40L(rMVA-CD40L) as described in Example 14. Where indicated, mice received200 μg anti-CD8 antibody intraperitoneally (IP). FIG. 19A: CD8 T cellresponses were measured. FIG. 19B: OVA-specific CD8 T cell responseswere measured. FIG. 19C represents overall survival.

FIGS. 20A-20C show that simultaneous targeting of two TAAs is moreefficient than targeting only one. C57BL/6 mice bearing palpable B16.OVAtumors were immunized IV either with PBS, MVA-OVA-CD40L, MVA-OVA-TRP2 orMVA-OVA-TRP2-CD40L as described in Example 15. Tumor growth was measuredat regular intervals and is displayed as (FIG. 20A) mean diameter ofindividual mice; and (FIG. 20B) mean volume. FIG. 20C: Overall survivalis shown.

FIGS. 21A-21G show increased T cell infiltration in the tumormicroenvironment (TME) after rMVA-CD40L immunization. C57BL/6 micebearing palpable B16.OVA tumors were immunized IV either with PBS,MVA-OVA (rMVA) or MVA-OVA-CD40L (rMVA-CD40L) as described in Example 16.Seven days later, mice were sacrificed. FIG. 21A: Frequency of CD8⁺ Tcells among CD45⁺ leukocytes in spleen, tumor-draining lymph nodes(TDLN) and tumor tissues; FIG. 21B: distribution of OVA₂₅₇₋₂₆₄-specificCD8⁺ T cells in different organs upon immunization; FIG. 21C: GMFI ofPD-1 and Lag3 on tumor-infiltrating OVA₂₅₇₋₂₆₄-specific CD8⁺ T cells;FIG. 21D: representative dot plots of tumor-infiltrating CD8⁺ T cellsshowing Ki67 and PD-1 expression; FIG. 21E: frequency oftumor-infiltrating Ki67⁺ CD8⁺ T cells and GMFI of PD-1; FIG. 21F:frequency of tumor-infiltrating regulatory T cells (Treg) among CD45⁺leukocytes; and FIG. 21G: frequency of PD-1^(high)- andPD-1^(neg)-tumor-infiltrating Treg.

FIGS. 22A-22D show persistence of TAA-specific CD8 T cells with a lessexhausted phenotype in the TME after rMVA-CD40L immunization. PurifiedOVA-specific TCR-transgenic CD8 T cells (OT-I) were IV transferred intoB16.OVA tumor bearers when tumors were palpable as described in Example17. When tumors reached at least 60 mm³ in volume animals were immunizedIV with MVA-BN, MVA-OVA (rMVA) or MVA-OVA-CD40L (rMVA-CD40L). 17 dayslater, mice were sacrificed and analyzed for: (FIG. 22A) Frequency ofCD8⁺ T cells among leukocytes in tumor tissues; (FIG. 22B) Frequency ofLag3⁺ PD1⁺ within CD8+ T cells (left graph); Frequency of Eomes+PD1⁺ Tcells within CD8+ T cells (right graph); (FIG. 22C) Presence ofOT-I-transgenic CD8⁺ T cells within the TME upon immunization; (FIG.22D) Frequency of Lag3⁺ PD1⁺ exhausted T cells within OT-I+CD8⁺ T cells(left graph); and Frequency of Eomes+PD1⁺ exhausted T cells withinOT-I+CD8⁺ T cells (right graph).

FIGS. 23A-23B show a long-term reduction of regulatory T cells (Treg) inthe TME after rMVA-CD40L immunization. Purified OVA-specificTCR-transgenic CD8 T cells (OT-I) were IV transferred into B16.OVA tumorbearers when tumors were palpable as described in Example 18. Whentumors reached at least 60 mm³ in volume animals were immunized IV withMVA-BN®, MVA-OVA (rMVA) or MVA-OVA-CD40L (rMVA-CD40L). 17 days later,mice were sacrificed for further analysis. FIG. 23A: Frequency of Foxp3⁺CD4⁺ Treg among CD4⁺ T cells in tumor tissues; FIG. 23B: Ratio of CD8⁺CD44⁺ effector T cells (Teff) to Foxp3⁺ CD4⁺ Tregs.

FIG. 24 shows lower serum levels of alanine aminotransferase (ALT) afterrMVA and rMVA-CD40L immunization compared to anti-CD40 immunoglobulininjection. C57BL/6 mice were injected IV either with PBS, MVA-OVA(rMVA), MVA-OVA-CD40L (rMVA-CD40L) or anti-CD40 (FGK4.5) as in Example19. Serum ALT concentration was analyzed one day after each immunizationby ELISA.

FIGS. 25A-25B show prolonged survival in a colon cancer model after IVrMVA-CD40L immunization. Balb/c mice bearing palpable CT26.HER2 tumorswere immunized IV with MVA-AH1A5-p15-Trp2-CD40L (rMVA-CD40L) or receivedPBS as described in Example 20. Tumor growth was measured at regularintervals. Shown are the tumor mean volume (FIG. 25A) and survival (FIG.25B).

FIGS. 26A-26B show increased anti-tumor effect of rMVA-CD40L incombination with anti-Trp1. C57BL/6 mice bearing palpable B16.OVA tumorswere immunized IV with MVA-OVA-CD40L (rMVA-CD40L) on day 8 as describedin Example 21. Where indicated 200 μg anti-Trp1 (clone TA99) wasinjected IP twice/week starting on day 5. Shown is the tumor mean volume(FIG. 26A) and overall survival (FIG. 26B).

FIGS. 27A-27D show transgene expression of MVA-HER2-Brachyury-CD40L.HeLa cells were left untreated (Mock; filled grey line) or infected withMVA-BN (filled black line) or MVA-HER2-Brachyury-CD40L (open black line)as described in Example 23. Then, (FIG. 27A) MVA, (FIG. 27B) HER2, (FIG.27C) Brachyury, and (FIG. 27D) CD40L protein expression was determinedby flow cytometry (see histograms).

FIG. 28 shows that Herceptin antibody (also known as trastuzumab) andPerjeta® antibody (also known as pertuzumab) anti-HER2 antibodies do notbind to the modified HER2 sequence described in Example 24. CT26 cellswere infected with MVA-HER2-Brachyury-CD40L at an MOI of 1. 24 hourslater, cells were incubated with 5 μg/ml of the HER2 antibodiesHerceptin® antibody, Perjeta® antibody, or 24D2 and analyzed by flowcytometry.

FIGS. 29A-29D show dose dependent and enhanced activation of human DCsby MVA-HER2-brachyury-CD40L as compared to MVA-HER2-brachyury.Monocyte-derived DCs were generated after enrichment of CD14′ monocytesfrom human PBMCs and cultured for 7 days in the presence of GM-CSF andIL-4 as described in Example 25. DCs were stimulated withMVA-HER2-brachyury or MVA-HER2-brachyury-CD40L. Expression of (FIG. 29A)CD40L; (FIG. 29B) CD86; and (FIG. 29C) MHC class II was measured by flowcytometry. FIG. 29D: The concentration of IL-12p70 was quantified.

FIGS. 30A and 30B show enhanced anti-tumor effect of IV (intravenous)MVA-HER2-Twist-CD40L immunization over IV (intravenous) MVA-CD40Limmunization in a HER2 positive colon carcinoma model. C57BL/6 micebearing palpable MC38.HER2 tumors were immunized (dotted line) eitherwith PBS, MVA-CD40L or MVA-HER2-Twist-CD40L IV as described in Example26. Tumor growth was measured at regular intervals. Shown are (FIG. 30A)tumor mean volume and (FIG. 30B) overall survival.

FIGS. 31A and 31B show enhanced anti-tumor effect of IVMVA-HER2-Twist-CD40L immunization over IV MVA-CD40L immunization in aHER2 positive colon carcinoma model. Balb/c mice bearing palpableCT26.HER2 tumors were immunized (dotted line) either with PBS, MVA-CD40Lor MVA-HER2-Twist-CD40L IV as described in Example 26. Tumor growth wasmeasured at regular intervals. Shown are (FIG. 31A) tumor mean volumeand (FIG. 31B) overall survival.

FIG. 32A shows increased infiltration of HER2-specific CD8⁺ T cellsproducing IFN-γ in the tumor microenvironment upon IV (intravenous)MVA-HER2-Twist-CD40L immunization. Balb/c mice bearing palpableCT26.HER2 tumors were immunized either with PBS or MVA-HER2-Twist-CD40LIV as described in Example 27. Seven days later, spleen andtumor-infiltrating CD8⁺ T cells isolated by magnetic cell sorting andcultured in the presence of HER2 peptide-loaded dendritic cells for 5hours. Graph shows percentage of CD44⁺ IFN-γ⁺ cells among CD8⁺ T cells.

FIGS. 33A and 33B show enhanced anti-tumor effect ofMVA-HER2-Twist-CD40L and Trastuzumab (anti-HER2). Balb/c mice bearinglarge, established 17-day-old CT26.HER2 tumors were immunized IV withMVA-HER2-Twist-CD40L (dotted line) as described in Example 28. Whereindicated 5 μg anti-HER2 or huIgG1 were injected IP (dashed line). Shownis the (FIG. 33A) tumor mean volume and (FIG. 33B) overall survival.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that both the foregoing Summary and the followingDetailed Description are exemplary and explanatory only and are notrestrictive of the invention, as claimed.

Antibody-dependent cellular cytotoxicity (ADCC) is a mechanism ofcell-mediated immune defense that enables the immune system to activelylyse and kill target cells that express an antigen or receptor that hasbeen bound by specific antibodies (see Hashimoto et al. (1983) J. InfDis. 148: 785-794). ADCC utilizes Natural Killer (NK) cells thatinteract with antibodies to lyse and kill the target cell (Id). Withthis in mind, over the past decade therapeutic monoclonal antibodieshave been developed which target tumor-associated antigens and functionto enhance ADCC and thus the ability to kill tumor cells (see, e.g.,Scott et al. (2012) Cancer Immun. 12: 14 and Kohrt et al. (2012)Immunother. 4: 511-27). While antibody therapies have shown efficacy inenhancing killing of tumor cells through ADCC, it has been shown thattumor cells can evade ADCC and the immune system through a variety ofmechanisms such as heterogeneity of tumor antigen expression (e.g.,downregulation of tumor antigen expression on the tumor cell surface),antibody stability, low antibody to receptor concentration, receptorsaturation, immune suppression for example through regulatory T cells,immune escape and NK cell dysfunction (Scott et al. (2012) Cancer Immun.12: 14). Some aspects of tumor cell evasion have been characterized asthe tumor cells entering an “equilibrium phase” with a patient's immunesystem, whereby tumor cells can remain in the body below the thresholdof conventional morphologic, cytogenetic or immune cell recognition(Bhatia et al. (2011) Cancer Microenvironment 4: 209-217).

To induce synergistic anti-tumor responses, the various pharmaceuticalcombinations of the present invention were developed. In severalaspects, the various pharmaceutical combinations induce both highlyeffective tumor specific killer T cells and natural killer (NK) cellsthat are able to kill tumor cells coated with antibody against tumorexpressed receptors through ADCC. In preferred aspects, as describedherein, intravenously injecting recombinant MVA encoding CD40L inducesan enhanced NK cell activation and drastically increased the kill ofantibody-coated tumor cells. This enhanced NK cell activation whencombined with the enhanced killer T cell response also induced by theMVA, is shown to act synergistically in therapeutic tumor vaccination.

In various additional aspects, the embodiments of the present inventioninduce an immune response that has enhanced and/or increased ability totarget and kill those tumor and cancer cells that evade a patient'simmune system. In further aspects, the embodiments of the presentinvention induce an immune response that enhances and/or increases theefficacy of both a patient's innate and adaptive immune responses. Beingable to enhance and/or increase both the innate and adaptive immuneresponses is particularly advantageous as the present invention cantarget and kill those tumor cells that evade and/or suppress a patient'sadaptive immune response as well as those tumor cells that evade and/orsuppress a patient's innate immune response.

In one embodiment, the present invention is a combination therapycomprising: a) an intravenous (IV) administration of a recombinant MVAthat comprises a nucleic acid encoding one or more heterologous tumorassociated antigens (TAA), and b) an antibody, wherein the antibodycomprises an Fe domain and is specific for an antigen expressed on atumor cell. In another embodiment, the combination therapy furthercomprises an IV administration of CD40L. In a preferred embodiment, theCD40L is encoded by the recombinant MVA.

Described and illustrated in the present application, the pharmaceuticalcombination and/or combination therapy of the present invention enhancesmultiple aspects of a cancer patient's immune response. In at least oneaspect, the pharmaceutical combination synergistically enhances both theinnate and adaptive immune responses to reduce tumor volume and increasesurvival of a cancer patient. One or more of the enhanced effects of thepharmaceutical combination and/or therapy are summarized as follows.

IV administration of recombinant MVA enhances NK cell response. In oneaspect, the present invention includes a recombinant MVA administeredintravenously to a subject, wherein the IV administration induces anenhanced innate immune response, more particularly an enhanced NK cellresponse in the subject as compared to a NK cell response induced by anon-IV administration of a recombinant MVA to the subject. Shown inFIGS. 1-9 and 13, IV administration of recombinant MVA induced a robustsystemic NK cell response in several compartments in both a single IVadministration and when administered intravenously as a homologousprime-boost, as compared to a non-IV administration.

Illustrated in FIGS. 1-6, the quality of the NK cell response wasenhanced as compared to a non-IV administration. The activation markerCD69 is increased in all organs analyzed (spleen, liver and lung). Theanti-apoptotic Bel-family member Bcl-X_(L), that enhances NK cellsurvival, co-stimulatory CD70 and the effector cytokine IFN-γ wereincreased both in spleen and lung. Expression of the activating NaturalKiller Group 2D (NKG2D) receptor was especially enhanced in liver andlung after IV compared to SC injection. NKG2D binds to ligands on tumorcells promoting their elimination (Garcia-Cuesta et al. (2015) Front.Immunol. 6: 284, reviewed in Spear et al. (2013) Cancer Immun. 13: 8).

IV administration of recombinant MVA encoding CD40L further enhances NKcell response. In another aspect of the present invention it wasdetermined that an IV administration of the CD40L antigen in addition tothe recombinant MVA further enhanced the NK cell response as compared toan IV administration of recombinant MVA alone. As illustrated in FIGS.1-9 and 13, a recombinant MVA encoding a CD40L antigen induced astronger NK cell response as compared to a recombinant MVA without CD40Lin both a single administration and when administered as a homologousprime boost. Further, the quality of the NK cell response was enhancedas compared to the IV administration of the recombinant MVA alone.Increased expression by NK cells of the effector cytokine IFN-γ wasobserved in all organs analyzed (FIGS. 1-6, spleen, liver, lung), aswell as expression of CD69 by NK cells in all organs analyzed (FIG. 5C).Moreover, FIG. 7 shows increased serum levels 6 hours after IVimmunization with rMVA-CD40L compared to recombinant MVA of IFN-γ and,more importantly, the NK activating cytokine IL-12p70, both in mice andNHPs. In addition, enhanced proliferation of NK cells, demonstrated bythe expression of Ki67, was observed not only systemically in mice (FIG.7B) but also in NHP peripheral blood (FIG. 6F). These results show thatIV immunization of rMVA-CD40L compared to rMVA improves NK cell qualityin several animal research models.

While recombinant MVA viruses have been previously administeredintravenously (see, e.g., WO2002/42480 and WO2014/037124), it waspreviously understood that recombinant MVA administration and treatmentwas associated with enhancement of an adaptive immune response, such asCD8 T cell responses. For example, in WO2002/42480, CTL responses weremeasured after immunizations using MVA were done either by IVadministration of 10⁷ pfu MVA-BN per mouse, or by subcutaneousadministration of 10⁷ pfu or 10⁸ pfu MVA-BN per mouse. In WO2014/037124,mice were intravenously inoculated with recombinant MVA and recombinantMVA encoding CD40L (see WO2014/037124). CTL responses were enhanced andit was determined that an increased immunogenicity of the recombinantMVA-CD40L was independent of CD4⁺ T cells but dependent upon CD40 in thehost.

In at least one aspect, the enhanced NK cell response seen by thepresent invention is unexpected as it was understood in the art thatMVA-induced NK cell activation was shown to be dependent on lymphnode-resident CD169-positive subcapsular sinus (SCS) macrophages aftersubcutaneous immunization (Garcia et al. (2012) Blood 120: 4744-50).

IV administration of recombinant MVA enhances ADCC of tumor cells. In afurther aspect, the present invention includes a recombinant MVAcomprising a nucleic acid encoding one or more heterologous antigens,wherein an IV administration to a subject enhances an ADCC response inthe subject as compared to an ADCC response from a non-IV administrationof a recombinant MVA to the subject. In a more specific aspect, theenhancement of the ADCC response resulting from an IV administration ofrecombinant MVA is characterized by an increase in NK cells' ability totarget and kill antibody-coated tumor cells. Illustrated in FIGS.10A-10D, cell lysis of tumor cells presenting a mouse anti-human HER2mAb or a mouse anti-human/mouse Trp1 mAb was increased for NK cellsactivated by an IV rMVA administration. Cell lysis of tumor cells waseven further increased for NK cells activated by a recombinant MVAtogether with CD40L as compared to rMVA without CD40L.

Illustrated in FIGS. 10D and 10E, the enhancement of an ADCC response inthe subject is additionally characterized as increasing the efficacy ofthe NK cell response such that the NK cells are able to target and killtumor cells whose antibody bound to tumor antigen (antibody-tumorantigen) concentration is lower. Being able to target and kill tumorcells with decreased concentrations of antibody-coated tumor antigen isparticularly advantageous, as one of the mechanisms by which tumor cellshave been shown to evade the immune system is through downregulation oftumor antigen expression (see Scott et al. (2012) Cancer Immun. 12: 14);which decreases the concentration of antibody-tumor antigen on tumorcells.

IV administration of recombinant MVA enhances NK cell killing of tumorcells having low MHC levels. In a further aspect, illustrated in FIG. 9,the enhanced NK cell activation and NK cell response results in anenhanced killing of tumor cells having low levels of MHC. This aspect isparticularly advantageous as NK cell killing of low MHC tumor cellsoccurs independently of ADCC, thereby adding an additional pathway toattack the tumor cells. This is additionally advantageous as many tumorsand/or cancers lower MHC expression levels in attempting to evade apatient's immune responses.

IV administration of recombinant MVA boosts NK cell activation andproliferation. In another aspect of the present invention, a recombinantMVA is administered in multiple boosts and results in increased NK cellactivation and proliferation demonstrated in FIG. 10A by means ofenhanced CD69 expression. FIG. 13B shows increased proliferativecapacity of blood NK cells by means of Ki67 expression 24 hours afterboost IV immunizations compared to no IV immunization. This effect wasobserved when a recombinant MVA prime immunization was boosted withrecombinant MVA, when a recombinant MVA-CD40L was boosted withrMVA-CD40L, or when rMVA prime immunization was boosted with rMVA-CD40L.

In at least one aspect of the present invention, the enhanced NK cellresponses resulting from the repeated recombinant MVA IV administrationand the recombinant MVA-CD40L were unexpected. It was unexpected toobserve increased NK cell activation and proliferation 24 hours afterboost IV immunizations in the absence of an IFN-α increase. Indeed, itwas understood that NK cell activation and priming in secondaryinfections and cancer is largely driven by IFN-α (see, e.g., Stackaruket al. (2013) Expert Rev. Vaccines 12(8): 875-84; and Muller et al.(2017) Front. Immunol. 8: 304). Unexpectedly, no increase in IFN-α serumlevels were observed 6 hours after rMVA hom, rMVA-CD40L hom orrMVA-CD40L het IV boost immunizations (FIG. 14C). Altogether, repeatedhomologous or heterologous IV immunizations with rMVA comprising anucleic acid encoding one or more heterologous antigens resulted inunexpected NK cell activation and proliferation independent of IFN-α.

IV administration of recombinant MVA encoding a heterologous antigenenhances a cancer patient's adaptive immune response. In another aspectof the present invention, there is a recombinant MVA comprising anucleic acid encoding one or more heterologous antigens, wherein an IVadministration to the subject enhances the subject's adaptive immuneresponse to the one or more heterologous antigens. In a preferredaspect, the recombinant MVA further encodes CD40L. Illustrated in FIGS.11-12, an IV administration of the recombinant MVA expressing aheterologous antigen produced a stronger CTL response as compared to asubcutaneous (SC) administration. Also, illustrated in the Figures, whenCD40L is included as part of the recombinant MVA, an IV administrationof the recombinant MVA antigen produced a stronger CTL response ascompared to an IV administration of a recombinant MVA without CD40L.

Further, illustrated in FIGS. 14-16, when recombinant MVA encoding aheterologous antigen and a CD40L were administered intravenously thequality of the T-cell response was enhanced after IV boostimmunizations. FIG. 15 shows increased MVA antigen, B8(FIG. 15B) andheterologous antigen, OVA (FIG. 15C) specific CD8 T cell expansionenhanced with boost IV immunizations. This effect is linked to thecytokine and chemokine expression pattern observed in FIG. 14, where theT cell activating cytokines CXCL10, IFN-γ, CXCL1, CCL4, CCL7, CCL2,CCL5, TNF-α, IL-12p70, and IL-18 are quantified in the serum after IVboosts. In line with this, FIGS. 16A and 16B show the expansion ofmemory T cells, a key feature of vaccines, after boost IV immunizations.The ratio of CD8 T cells specific for the heterologous antigen to MVAantigen-specific CD8 T cells increased with each immunization (FIG.15D).

Prior to the present invention, it was understood that CD40L encoded byrecombinant MVA can substitute for CD4 T cell help (Lauterbach et al.(2013) Front. Immunol. 4: 251). Further, no effect of recombinantMVA-encoded CD40L on CD4 T cells was known. Unexpectedly, we sawexpansion of memory CD4⁺ T cells 25 days after prime immunization (FIG.16B), which corresponds with 4 days after boost IV immunization withrMVA-CD40L (rMVA-CD40L hom and rMVA-CD40L het) (Day 21, see Table 1).This fact is supported by the increased IL-22 production, an importantcytokine indicative of T helper cell responses, quantified 6 hours afterboost IV immunization in MVA-CD40L hom and MVA-CD40L het groups (FIG.14D). This unexpected observation is relevant for the maintenance ofmemory responses by rMVA-CD40L. Furthermore, CD4 T cells can supporttumor-specific CD8 T cells at the tumor site, avoid activation-inducedcell death and also become cytotoxic themselves (reviewed in Kennedy andCelis (2008) Immunol. Rev. 222: 129-44; Knutson and Disis (2005) Curr.Drug Targets Immune Endocr. Metabol. Disord 5: 365-71). These resultsare unexpected because other viral vectors, such as Adenovirus andHerpes Simplex Virus, induce vector-specific immunity that impede theinduction of immune responses to the vaccine-encoded antigens upon boostimmunization (Lauterbach et al. (2005) J. Gen. Virol. 86: 2401-10; Pineet al. (2011) PLoS One 6: e18526).

IV Administration of recombinant MVA at the same as or afteradministration of an antibody increases effectiveness of tumor cellkilling. In still further aspects, the present disclosure provides forone or more regimens for administration of the pharmaceuticalcombination of the present invention to a subject. In at least oneaspect, the regimens of the present invention increase the effectivenessof the pharmaceutical combination and/or therapy to enhanceADCC-mediated killing of the tumor cells. In one embodiment, a regimenof administration of the pharmaceutical combination comprises a)administering an antibody as described herein, and b) at the same timeor after the antibody administration, intravenously administering arecombinant MVA of the present invention to the subject.

In one advantageous aspect of the present invention, administering therecombinant MVA at the same time or after the antibody enablesadministered antibody to bind tumor cells at the same time or prior toenhancement of the NK cell response that results from administering therecombinant MVA. Accordingly, in an exemplary first step, an antibody isadministered resulting in the antibody binding to the tumor or diseaseinfected cells. In an exemplary second step, recombinant MVA isintravenously administered which, as described herein, enhances andincreases the subject's NK cell response. The enhanced NK cell responsethen aggressively targets and kills tumor cells having the boundantibody.

In other aspects, the pharmaceutical combination of the presentinvention is administered as part of a homologous and/or heterologousprime-boost regimen. Illustrated in FIGS. 11-16, a homologous and/orheterologous prime boost regimen prolongs and reactivates enhanced NKcell responses as well as increases a subject's specific CD8 and CD4 Tcell responses.

IV administration of recombinant MVA enhances anti-tumor effects. Inanother aspect of the present invention, there is a recombinant MVAcomprising a nucleic acid encoding one or more heterologous antigens,wherein an IV administration to the subject results in an increase inthe survival rate of the subject as well as a reduction in the overalltumor volume, as compared to a non-IV administration. In a preferredaspect, the recombinant MVA further encodes CD40L. Illustrated in FIGS.17 and 18, an IV administration of the recombinant MVA resulted in agreater overall survival rate and a greater reduction in tumor volume ora longer control of tumor growth as compared to a SC administration.Also illustrated, CD40L included as part of the recombinant MVA producedan improved overall survival and tumor reduction as compared to an IVadministration of a recombinant MVA without CD40L. Illustrated in FIG.19, CD40L included as part of the recombinant MVA increased total CD8and antigen (OVA)-specific T cell accumulation in peripheral blood oftumor bearers compared to an IV administration of a recombinant MVAwithout CD40L. In addition, CD8 T cells are critical mediators ofrMVA-CD40L anti-tumor effect, since antibody-mediated depletion of CD8cells results in lack of prolonged overall survival.

IV administration of MVA encoding for two heterologous antigensincreases anti-tumor efficacy. In another aspect of the presentinvention illustrated in FIG. 291, IV immunization utilizing MVAencoding for two heterologous antigens (OVA and TRP2) induces prolongedtumor growth control and increases overall survival. CD40L included aspart of the recombinant MVA encoding for two heterologous antigensenhances the anti-tumor response compared to an IV administration of arecombinant MVA with CD40L and only one heterologous antigen. Sincecancers utilize diverse mechanisms of immune escape as downregulation oftumor antigens (Jensen et al. (2012) Cancer 118: 2476-85), encoding twoor more tumor antigens in the vaccine limits the development of escapemechanisms to the anti-tumor immune response.

IV administration of MVA reduces a tumor's immunosuppressive effects.Illustrated in FIGS. 29 and 32, intravenously administered recombinantMVA encoding a heterologous antigen and optionally a CD40L, inducedinfiltration of CD8⁺ T cells in the tumor and reduced multipleimmunosuppressive effects typically employed by tumors to evade theimmune system. In addition to increased endogenous CD8⁺ cells within thetumors upon recombinant MVA with or without CD40L challenge, antigen(OVA)-specific T cells were increased in spleen and tumors upon IVadministration of a recombinant MVA with CD40L compared to MVA withoutCD40L. Moreover, tumor infiltrating T lymphocytes expressed lessimmunosuppressive surface molecules PD-1 and Lag3 when checked inendogenous T cells (FIG. 22C-E, 23B) or transferred antigen(OVA)-specific T cells (FIG. 21 D) and were proliferating in the tumormicroenvironment. Reduced PD-I expression on tumor-infiltrating T cellsafter rMVA and rMVA-CD40L immunization was unexpected because type I andII interferons, which are induced by both vectors (FIGS. 14C and E), areknown inducer of PD-1 and PD-L1 expression (reviewed by Dong et al.(2017) Oncotarget 8: 2171-86). Unexpectedly, immunosuppressive Tregulatory cell (Treg) numbers in the tumor microenvironment weredecreased (22F, 23A) when recombinant MVA encoding a heterologousantigen and optionally a CD40L, resulting in an enhanced effector T cellto Treg ratio in the tumor microenvironment (FIG. 23B). Reducedexpression of immune-dampening molecules such as PD-I and Lag3 as wellas reduced numbers of Tregs in the tumor tissue correlate with theenhanced anti-tumor effects seen after rMVA and rMVA-CD40L immunization.

The pharmaceutical combination of the present invention reduces tumorburden and increases survival rate in cancer patients. In variousembodiments, the pharmaceutical combination includes a) an IVadministration of a recombinant MVA encoding a heterologous TAA andoptionally CD40L and b) an administration of an antibody. Shown in FIGS.26 and 33, the pharmaceutical combination resulted in a reduction intumor volume and an increase in overall survival rate.

In at least one aspect, the enhanced anti-tumor effects of thepharmaceutical combination (e.g., reduced tumor volume and/or increasedsurvival rate) is achieved from the synergistic combining of theindividual enhancements of the innate and adaptive T cell responsesdescribed herein. In one exemplary embodiment, these individualenhancements include one or more of those listed above, e.g., anenhanced innate (e.g., NK cell) response, enhanced ADCC mediated killingof tumor cells, enhanced NK cell killing of tumor cells having lower MHCclass I levels, and an enhanced adaptive T cell response. Furthermore,the one or more dosing regimens of the present invention further improveand enable a patient's immune system to kill tumor cells over a periodof time.

IV Administration of MVA-HER2-Brachyury plus anti-HER2 antibody. In oneadvantageous embodiment of the invention, the pharmaceutical combinationcomprises a) an intravenous administration of a recombinant MVA encodingHER2 and Brachyury antigens and optionally a CD40L and b) anadministration of an anti-HER2 antibody. This embodiment induces theenhanced immune response described herein and additionally focuses tumorcell killing on HER2 and Brachyury expressing tumor cells. In additionaladvantageous aspects, the HER2 antigen comprises one more modificationsthat further increase the efficacy of the combination therapy of thepresent invention. Shown in FIG. 29, the rMVA-HER2-Brachyury and therMVA-HER2-Brachyury-CD40L showed activation of Dendritic Cells (DCs). DCactivation was enhanced by rMVA-HER2-Brachyury-CD40L as compared torMVA-HER2-Brachyury. Shown in FIG. 33, administration of thepharmaceutical combination described in the present invention resultedin a significant increase in tumor reduction and overall survival rateof the subjects.

In one specific embodiment, the present invention is directed andtailored to treat cancer patients with HER2 expressing malignancies suchas HER2 positive breast or gastric cancers which are treated with HER2binding antibodies. The recombinant MVA encoding HER2 induces highlyeffective killer T cells against the HER2 expressing tumor cells whilethe Brachyury transgene encoded by the recombinant MVA induces highlyeffective killer T cells against Brachyury tumor cells that have thepotential to be metastatic.

Definitions

As used herein, the singular forms “a,” “an,” and “the,” include pluralreferences unless the context clearly indicates otherwise. Thus, forexample, reference to “a nucleic acid” includes one or more of thenucleic acid and reference to “the method” includes reference toequivalent steps and methods known to those of ordinary skill in the artthat could be modified or substituted for the methods described herein.

Unless otherwise indicated, the term “at least” preceding a series ofelements is to be understood to refer to every element in the series.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the present invention.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise,” and variations such as“comprises” and “comprising,” will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integer or step. Whenused herein the term “comprising” can be substituted with the term“containing” or “including” or sometimes when used herein with the term“having.” Any of the aforementioned terms (comprising, containing,including, having), though less preferred, whenever used herein in thecontext of an aspect or embodiment of the present invention can besubstituted with the term “consisting of. When used herein “consistingof” excludes any element, step, or ingredient not specified in the claimelement. When used herein, “consisting essentially of” does not excludematerials or steps that do not materially affect the basic and novelcharacteristics of the claim.

As used herein, the conjunctive term “and/or” between multiple recitedelements is understood as encompassing both individual and combinedoptions. For instance, where two elements are conjoined by “and/or,” afirst option refers to the applicability of the first element withoutthe second. A second option refers to the applicability of the secondelement without the first. A third option refers to the applicability ofthe first and second elements together. Any one of these options isunderstood to fall within the meaning, and therefore satisfy therequirement of the term “and/or” as used herein. Concurrentapplicability of more than one of the options is also understood to fallwithin the meaning, and therefore satisfy the requirement of the term“and/or.”

“Mutation” described herein is as defined herein any a modification to anucleic acid or amino acid, such as deletions, additions, insertions,and/or substitutions.

A “host cell” as used herein is a cell that has been introduced with aforeign molecule, virus, or microorganism. In one non-limiting example,as described herein, a cell of a suitable cell culture as, e.g., CEFcells, can be infected with a poxvirus or, in other alternativeembodiments, with a plasmid vector comprising a foreign or heterologousgene. Thus, a suitable host cell and cell cultures serve as a host topoxvirus and/or foreign or heterologous gene.

“Percent (%) sequence homology or identity” with respect to nucleic acidsequences described herein is defined as the percentage of nucleotidesin a candidate sequence that are identical with the nucleotides in thereference sequence (i.e., the nucleic acid sequence from which it isderived), after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Alignment for purposes of determining percent nucleotidesequence identity or homology can be achieved in various ways that arewithin the skill in the art, for example, using publicly availablecomputer software such as BLAST, ALIGN, or Megalign (DNASTAR) software.Those skilled in the art can determine appropriate parameters formeasuring alignment, including any algorithms needed to achieve maximumalignment over the full length of the sequences being compared.

For example, an appropriate alignment for nucleic acid sequences isprovided by the local homology algorithm of Smith and Waterman ((1981)Advances in Applied Mathematics 2: 482-489). This algorithm can beapplied to amino acid sequences by using the scoring matrix developed byDayhoff (Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5suppl. 3: 353-358, National Biomedical Research Foundation, Washington,D.C., USA), and normalized by Gribskov ((1986) Nucl. Acids Res. 14(6):6745-6763). An exemplary implementation of this algorithm to determinepercent identity of a sequence is provided by the Genetics ComputerGroup (Madison, Wis.) in the “BestFit” utility application. The defaultparameters for this method are described in the Wisconsin SequenceAnalysis Package Program Manual, Version 8 (1995) (available fromGenetics Computer Group, Madison, Wis.). A preferred method ofestablishing percent identity in the context of the present invention isto use the MPSRCH package of programs copyrighted by the University ofEdinburgh, developed by Collins and Sturrok, and distributed byIntelliGenetics, Inc. (Mountain View, Calif.). From this suite ofpackages the Smith-Waterman algorithm can be employed where defaultparameters are used for the scoring table (for example, gap open penaltyof 12, gap extension penalty of one, and a gap of six). From the datagenerated, the “Match” value reflects “sequence identity.” Othersuitable programs for calculating the percent identity or similaritybetween sequences are generally known in the art, for example, anotheralignment program is BLAST, used with default parameters. For example,BLASTN and BLASTP can be used using the following default parameters:genetic code=standard; filter=none; strand=both; cutoff-60; expect=10;Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE;Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+Swiss protein+Spupdate+PIR. Details of these programs canbe found at the following internet address: blast.ncbi.nlm.nih.gov/.

The term “prime-boost vaccination” or “prime-boost regimen” refers to avaccination strategy or regimen using a first priming injection of avaccine targeting a specific antigen followed at intervals by one ormore boosting injections of the same vaccine. Prime-boost vaccinationmay be homologous or heterologous. A homologous prime-boost vaccinationuses a vaccine comprising the same antigen and vector for both thepriming injection and the one or more boosting injections. Aheterologous prime-boost vaccination uses a vaccine comprising the sameantigen for both the priming injection and the one or more boostinginjections but different vectors for the priming injection and the oneor more boosting injections. For example, a homologous prime-boostvaccination may use a recombinant poxvirus comprising nucleic acidsexpressing one or more antigens for the priming injection and the samerecombinant poxvirus expressing one or more antigens for the one or moreboosting injections. In contrast, a heterologous prime-boost vaccinationmay use a recombinant poxvirus comprising nucleic acids expressing oneor more antigens for the priming injection and a different recombinantpoxvirus expressing one or more antigens for the one or more boostinginjections.

The term “recombinant” means a polynucleotide, virus or vector ofsemisynthetic, or synthetic origin which either does not occur in natureor is linked to another polynucleotide in an arrangement not found innature.

As used herein, reducing or a reduction in tumor volume can becharacterized as a reduction in tumor volume and/or size but can also becharacterized in terms of clinical trial endpoints understood in theart. Some exemplary clinical trial endpoints associated with a reductionin tumor volume and/or size can include, but are not limited to,Response Rate (RR), Objective response rate (ORR), and so forth.

As used herein an increase in survival rate can be characterized as anincrease in survival of a cancer patient, but can also be characterizedin terms of clinical trial endpoints understood in the art. Someexemplary clinical trial endpoints associated with an increase insurvival rate include, but are not limited to, overall survival rate(ORR), Progression free survival (PFS) and so forth.

As used herein, a “transgene” or “heterologous” gene is understood to bea nucleic acid or amino acid sequence which is not present in thewild-type poxviral genome (e.g., Vaccinia, Fowlpox, or MVA). The skilledperson understands that a “transgene” or “heterologous gene”, whenpresent in a poxvirus, such as Vaccinia Virus, is to be incorporatedinto the poxviral genome in such a way that, following administration ofthe recombinant poxvirus to a host cell, it is expressed as thecorresponding heterologous gene product, i.e., as the “heterologousantigen” and\or “heterologous protein.” Expression is normally achievedby operatively linking the heterologous gene to regulatory elements thatallow expression in the poxvirus-infected cell. Preferably, theregulatory elements include a natural or synthetic poxviral promoter.

A “vector” refers to a recombinant DNA or RNA plasmid or virus that cancomprise a heterologous polynucleotide. The heterologous polynucleotidemay comprise a sequence of interest for purposes of prevention ortherapy, and may optionally be in the form of an expression cassette. Asused herein, a vector needs not be capable of replication in theultimate target cell or subject. The term includes cloning vectors andviral vectors.

Pharmaceutical Combinations and Methods

In various embodiments, the present invention includes a pharmaceuticalcombination for treating a cancer patient by reducing tumor volumeand/or increasing survival in the cancer patient. The pharmaceuticalcombination comprises a recombinant MVA comprising a first nucleic acidencoding a first heterologous tumor-associated antigen (TAA) that whenadministered intravenously induces both an enhanced Natural Killer (NK)cell response and an enhanced T cell response as compared to a NK cellresponse and a T cell response induced by a non-intravenousadministration of a recombinant MVA virus comprising a nucleic acidencoding a heterologous tumor-associated antigen.

Enhanced NK Cell Response

In one aspect, an enhanced NK cell response according to the presentdisclosure is characterized by one or more of the following: 1) anincrease in NK cell frequency, 2) an increase in NK cell activation,and/or 3) an increase in NK cell proliferation. Thus, whether an NK cellresponse is enhanced in accordance with the present disclosure can bedetermined by measuring the expression of one or more cytokines whichare indicative of an increased NK cell frequency, increased NK cellactivation, and/or increased NK cell proliferation. Exemplary markersthat are useful in measuring NK cell frequency and/or activity includeone or more of: NKp46, IFN-γ, CD69, CD70, NKG2D, FasL, granzyme B, CD56,and/or Bcl-X_(L). Exemplary markers that are useful in measuring NK cellactivation include one or more of IFN-γ, CD69, CD70, NKG2D, FasL,granzyme B and/or Bcl-X_(L). Exemplary markers that are useful inmeasuring NK cell proliferation include: Ki67. These molecules and themeasurement thereof are validated assays that are understood in the artand can be carried out according to known techniques (see, e.g., Borregoet al. (1999) Immunology 97: 159-65; Fogel et al. (2013) J. Immunol.190: 6269-76). Additionally, assays for measuring the molecules can befound in Examples 1-3, and 9 of the present disclosure. At least in oneaspect, 1) an increase in NK cell frequency can be defined as at least a2-fold increase in CD3-NKp46⁺ cells compared to pre-treatment/baseline;2) an increase in NK cell activation can be defined as at least a 2-foldincrease in IFN-γ, CD69, CD70, NKG2D, FasL, granzyme B and/or Bcl-X_(L)expression compared to pre-treatment/baseline expression; and/or 3) anincrease in NK cell proliferation is defined as at least a 1.5 foldincrease in Ki67 expression compared to pre-treatment/baselineexpression.

In a more preferred aspect, an “enhanced NK cell response” as used inthe present disclosure is characterized by an increase in NK cellmediated tumor cell killing. NK cell mediated tumor cell killing can beanalyzed by measuring release of Lactate Dehydrogenase (LDH) into thecell culture medium, as shown in Example 5. Thus, within the context ofthe present invention, whether or not a pharmaceutical combination“enhances an NK cell response” can be characterized by the existence ofan increase in NK cell mediated tumor cell killing. NK cell mediatedtumor cell killing can be analyzed by measuring the release of LactateDehydrogenase as seen in Example 5. Assays associated with LDH and themeasurement thereof are validated and understood in the art. At leastone aspect, the increase in NK cell mediated tumor cell killing can bedefined as at least a 2-fold increase in tumor cell killing.

Enhanced ADCC Response

In at least one aspect, the enhanced NK cell response induced by thepresent invention results in an enhanced ADCC response. An “enhancedADCC response” according to the present invention is characterized by anincrease in innate immune effector cells' ability to target and killtumor cells coated by antibodies. In the context of the presentdisclosure an innate immune effector can include, but is not limited to,NK cells, macrophages, neutrophils, basophils, eosinophils, mast cells,dendritic cells and so forth. In a more preferred aspect, an innateimmune effector is an NK cell. Assays that are useful for measuring aneffector cell's ability to kill tumor cells include ex vivoEffector:Target killing assays, where “effector” are isolated effectorcells and “target” are tumor cells in the presence of antibodies (seeYamashita et al. (2016) Sci. Rep. 6: 19772 and Broussas et al. (2013)Methods Mol. Biol. 988: 305-17). The ex vivo Effector:Target killingassays are validated and understood in the art. Additionally, assays formeasuring an effector cell's ability to kill tumor cells can be found inExample 5. Thus, in one aspect, within the context of the presentapplication, whether or not an enhanced innate immune response,including an enhanced NK cell response, enhances ADCC can becharacterized by measuring levels of tumor cell killing through one ormore of: 1) using different ratios of effector cells per coated targetcell (tumor cell coated with antibody); and 2) using a constant ratio ofeffector cells and one or more concentrations of antibodies e.g., lowerconcentrations of antibody bound to tumor antigen are compared to levelsof tumor cell killing at higher concentrations.

In a preferred aspect “enhances ADCC response” according to the presentdisclosure is characterized by an increase in NK cells' ability totarget and kill tumor cells coated by antibodies. Assays that are usefulfor measuring an NK cell's ability to kill tumor cells include ex vivoEffector:Target killing assays, where “effector” are isolated NK cellsand “target” are tumor cells in the presence of antibodies.Additionally, assays for measuring a NK cell's ability to kill tumorcells can be found in Example 5. Thus, in one aspect, within the contextof the present application, whether or not an NK cell response enhancesADCC can be characterized by measuring levels of tumor cell killing: 1)using different ratios of NK cells per coated target cell (tumor cellcoated with antibody); and/or 2) using a constant ratio of NK cells andone or more concentrations of antibodies, e.g., lower concentrations ofantibody bound to tumor antigen are compared to levels of tumor cellkilling at higher concentrations.

Stronger NK Cell Mediated Toxicity

In additional aspects, the enhanced NK cell response results in anincreased ability of the innate immune effectors, such as NK cells, toattack and kill tumor cells having low MHC expression levels. In oneaspect killing tumor cells having low MHC levels can be characterized byinnate immune effectors, e.g., NK cells, having a stronger NK cellmediated toxicity, meaning NK cells are more efficiently killing tumorcells. NK cells having a stronger NK cell mediated toxicity can bemeasured by an increased number of tumor cells killed per NK cells. Thiscan be determined through the use of an effector: target killing assay.Such assays are validated and known in the art and demonstrated by thepresent application in at least Example 4 and FIG. 9.

Enhanced Innate Immune Effector Cells

In additional aspects, an IV administration of the recombinantpoxviruses of the present application not only enhances the NK cellresponse, but also enhances all aspects of the innate immune response.Enhancement of the innate immune response can be characterized as anincrease in innate effector cell: 1) frequency, 2) activation, and/or 3)proliferation. The innate effector cells can include NK cells, innatelymphoid cells (ILCs), macrophages, neutrophils, basophils, eosinophils,mast cells, monocytes, and/or dendritic cells. In various aspects,whether an innate immune response is enhanced in accordance with thepresent application can be determined by measuring one or more of theexpression of cytokines and/or activation markers associated with thedescribed effector cells of the innate response. These cytokines andactivation markers and the measurement thereof are validated andunderstood in the art and can be carried out according to knowntechniques (see Borrego et al. (1999) Immunology 97: 159-65; Fogel etal. (2013) J. Immunol. 190: 6269-76).

Enhanced T Cell Response

In accordance with the present application, an “enhanced T cellresponse” is characterized by one or more of the following: 1) anincrease in frequency of CD8 T cells; 2) an increase in CD8 T cellactivation; and/or 3) an increase in CD8 T cell proliferation. Thus,whether a T cell response is enhanced in accordance with the presentapplication can be determined by measuring the expression of one or morecytokines which are indicative of 1) an increase in CD8 T cell frequency2) an increase in CD8 T cell activation; and/or 3) an increase CD8 Tcell proliferation. Exemplary markers that are useful in measuring CD8 Tcell frequency, activation, and proliferation include CD3, CD8, IFN-γ,TNF-α, IL-2, CD69 and/or CD44, and Ki67, respectively. Measuring antigenspecific T cell frequency can also be measured by ELIspot or MHCMultimers such as pentamers or dextramers as shown in FIGS. 11, 12, 15,19, and 21. Such measurements and assays are validated and understood inthe art.

In one aspect, an increase in CD8 T cell frequency is characterized byan at least a 2-fold increase in IFN-γ and/or dextramer⁺ CD8 T cellscompared to pre-treatment/baseline. An increase in CD8 T cell activationis characterized as at least a 2-fold increase in CD69 and/or CD44expression compared to pre-treatment/baseline expression. An increase inCD8 T cell proliferation is characterized as at least a 2-fold increasein Ki67 expression compared to pre-treatment/baseline expression.

In an alternative aspect, an enhanced T cell response is characterizedby an increase in CD8 T cell expression of effector cytokines and/or anincrease of cytotoxic effector functions. An increase in expression ofeffector cytokines can be measured by expression of one or more ofIFN-γ, TNF-α, and/or IL-2 compared to pre-treatment/baseline. Anincrease in cytotoxic effector functions can be measured by expressionof one or more of CD107a, granzyme B, and/or perforin and/orantigen-specific killing of target cells.

The assays, cytokines, markers, and molecules described herein and themeasurement thereof are validated and understood in the art and can becarried out according to known techniques. Additionally, assays formeasuring the T cells responses can be found in Examples 6 and 7,wherein T cell responses were analyzed.

The enhanced T cell response realized by the present invention isparticularly advantageous in combination with the enhanced NK cellresponse, as the enhanced T cells effectively target and kill thosetumor cells that have evaded and/or survived past the initial innateimmune responses in the cancer patient. Furthermore, antibody treatmentcan enhance MHC class I presentation of TAAs, resulting in highersusceptibility of TAA-expressing tumors to lysis by TAA-specific T cells(Kono et al. (2004) Clin. Cancer Res. 10: 2538-44).

In additional embodiments, the pharmaceutical combination furthercomprises an antibody, wherein the antibody is specific to an antigenthat is expressed or overexpressed on the cell membrane, preferably anouter cell membrane of a tumor cell. It is contemplated that theantibody can be any antibody as described by the present disclosure. Inpreferred embodiments, the antibody comprises an Fc domain.

In additional embodiments, there is a method for reducing tumor volumeand/or increasing survival in a cancer patient. The method comprises a)intravenously administering to the cancer patient a recombinant MVAcomprising a first nucleic acid encoding a first heterologoustumor-associated antigen (TAA) that when administered intravenouslyinduces both an enhanced innate immune response and an enhanced T cellresponse as compared to an innate immune response and a T cell responseinduced by a non-intravenous administration of a recombinant MVA viruscomprising a nucleic acid encoding a heterologous tumor-associatedantigen; and b) administering to the cancer patient an antibody, whereinthe antibody comprises an Fc domain and is specific to an antigen thatis expressed on the cell membrane of a tumor cell, whereinadministration of a) and b) to the cancer patient reduces tumor size inthe cancer patient and/or increases survival of the cancer patient ascompared to a non-intravenous administration of a) or an administrationof b) alone. In a more preferred embodiment, the enhanced innate immuneresponse comprises an enhanced NK cell response as described herein.

In another embodiment, there is a method for enhancing antibody therapyin a patient, the method comprising a) administering to the cancerpatient an antibody, wherein the antibody comprises an Fc domain and isspecific to an antigen that is expressed on the cell membrane of a tumorcell, and wherein administering the antibody induces ADCC in thepatient; and b) intravenously administering to the cancer patient arecombinant MVA comprising a first nucleic acid encoding a firstheterologous tumor-associated antigen (TAA) that induces both anenhanced innate immune response and an enhanced T cell response in thepatient, wherein the enhanced innate immune response enhances the ADCCin the patient, as compared to a non-intravenously administeredcombination of a) and b) or a) and b) alone. In a more preferredembodiment, the enhanced innate immune response comprises an enhanced NKcell response as described herein.

In still another embodiment there is a method for increasing theeffectiveness of antibody therapy in a cancer patient, the methodcomprising administering the pharmaceutical combination of the presentinvention, wherein administering the combination to the patientdecreases the antibody concentration needed for NK cell-mediatedtoxicity in tumor cells. In at least one advantageous aspect, decreasingantibody concentration needed for NK cell mediated toxicity enables theenhanced NK cell response to kill tumor cells that have decreasedextracellular antigen expression in order to evade destruction by boundantibody targets. Additionally, decreasing needed antibody concentrationcan enable treatment with decreasing amounts of antibody.

In the context of the present application, decreasing the antibodyconcentration needed for NK cell mediated toxicity can be characterizedas a decrease in antibody concentration needed for tumor cell killing. Adecrease in antibody concentration needed for tumor cell killing can bemeasured by an ex vivo Effector: target killing assays, where “effector”are isolated NK cells and “target” are tumor cells in the presence ofantibodies. Additionally, assays for measuring include measuring levelsof tumor cell killing by for example, 1) using different ratios of NKcells per coated target cell (tumor cell coated with antibody); and/or2) using a constant ratio of NK cells and one or more concentrations ofantibodies e.g., lower concentrations of antibody bound to tumor antigenare compared to levels of tumor cell killing at higher concentrations.Such assays are known in the art and also presently described in Example5 and 31 of the present application.

In yet additional embodiments, the pharmaceutical combination andmethods described herein are for treating a human cancer patient. Inpreferred embodiments, the cancer patient is suffering from and/or isdiagnosed with a cancer selected from the group consisting of: breastcancer, lung cancer, head and neck cancer, thyroid, melanoma, gastriccancer, bladder cancer, kidney cancer, liver cancer, melanoma,pancreatic cancer, prostate cancer, ovarian cancer, urothelial,cervical, or colorectal cancer.

In still additional preferred embodiments, the pharmaceuticalcombinations and methods of the present invention each comprise CD40L.Preferably the CD40L is encoded by the recombinant MVA.

Exemplary Tumor-Associated Antigens

In certain preferred embodiments, the first and/or second TumorAssociated antigen (TAA) includes but is not limited to HER2, PSA, PAP,CEA, MUC-1, survivin, TYRP1, TYRP2, or Brachyury alone or incombinations. Such exemplary combination may include HER2 and Brachyury,CEA and MUC-1, or PAP and PSA.

Numerous TAAs are known in the art. Exemplary TAAs include, but are notlimited to, 5 alpha reductase, alpha-fetoprotein, AM-1, APC, April,BAGE, beta-catenin, Bcl12, bcr-abl, Brachyury, CA-125, CASP-8/FLICE,Cathepsins, CD19, CD20, CD21, CD23, CD22, CD33 CD35, CD44, CD45, CD46,CD5, CD52, CD55, CD59, CDC27, CDK4, CEA, c-myc, Cox-2, DCC, DcR3, E6/E7,CGFR, EMBP, Dna78, farnesyl transferase, FGF8b, FGF8a, FLK-1/KDR, folicacid receptor, G250, GAGE-family, gastrin 17, gastrin-releasing hormone,GD2/GD3/GM2, GnRH, GnTV, GP1, gp100/Pmel17, gp-100-in4, gp15,gp75/TRP-1, hCG, heparanase, HER2/neu, HMTV, Hsp70, hTERT, IGFR1,IL-13R, iNOS, Ki67, KIAA0205, K-ras, H-ras, N-ras, KSA, LKLR-FUT,MAGE-family, mammaglobin, MAP17, melan-A/MART-1, mesothelin, MIC A/B,MT-MMPs, mucin, 25 NY-ESO-1, osteonectin, p15, P170/MDR1, p53,p97/melanotransferrin, PAI-1, PDGF, uPA, PRAME, probasin,progenipoietin, PSA, PSM, RAGE-1, Rb, RCAS1, SART-1, SSX-family, STAT3,STn, TAG-72, TGF-alpha, TGF-beta, Thymosin-beta-15, TNF-alpha, TYRP-,TYRP-2, tyrosinase, VEGF, ZAG, p16INK4, and glutathione-S-transferase.

A preferred PSA antigen comprises the amino acid change of isoleucine toleucine at position 155, as found in U.S. Pat. No. 7,247,615, which isincorporated herein by reference.

In more preferred embodiments of present invention, the first and/orsecond heterologous TAA are selected from HER2 and Brachyury. In evenmore preferred embodiments, the first and/or second heterologous TAA areselected from the synthetic HER2 and Brachyury proteins as describedherein.

In additional preferred embodiments, the first and/or secondheterologous TAA comprise one or more mutations. In at least oneembodiment, the one or more mutations comprise mutations that preventthe antibodies of the present disclosure from binding to the firstand/or second heterologous TAA. In additional embodiments, the one ormore mutations comprise mutations that prevent the first and/or secondTAA from performing the one or more normal cellular functions of theTAA. Exemplary embodiments of the one or more mutations are described bythe synthetic HER2 and synthetic Brachyury proteins of the presentinvention.

Synthetic HER2 Proteins

With the development of the present invention, the inventors determinedthat one or more modifications of the HER2 antigens and/or nucleic acidsencoding the HER2 antigens as described herein increase the efficacy ofthe combination therapy.

Accordingly, in various embodiments the present invention includes anucleic acid encoding a HER2 antigen selected from HER2v1 and HER2v2.HER2v1 and HER2v2 comprise SEQ ID NO: 1 and SEQ ID NO: 13, respectively.

In at least one specific aspect, the nucleic acid encoded by SEQ ID NO:1and/or 13 is particularly advantageous as SEQ ID NO: 1 and/or 13 isconfigured to function synergistically with antibodies to the HER2antigen when administered as part of the combination therapy.

In one exemplary embodiment of the present invention, the HER2antibodies comprise those antibodies that are approved for treatment ofa HER2 expressing cancer, or in more specific embodiment, a HER2expressing breast cancer. Two humanized monoclonal antibodies targetingHER2 have been developed and approved for treatment of HER2-expressingbreast cancer. Both antibodies bind at different sites in theextracellular domain of HER2. Trastuzumab (branded as Herceptin® andhaving Herzuma and ABP 980 as biosimilars) binding results in signaltransduction blockade and prevention of HER2 cleavage. In contrast,Pertuzumab (Perjeta®) sterically blocks HER2 dimerization with other EGFreceptors and blocks ligand-activated signaling. Both Trastuzumab andPertuzumab provide a dual blockade of HER2-driven signaling pathways andin addition have the ability to mediate ADCC against breast cancer.Thus, in various specific embodiments of the present invention, theantibodies of the present invention comprise Trastuzumab, Herzuma, ABP980, and/or Pertuzumab.

Synthetic HER2 v

In one or more embodiments, the synthetic HER2 protein is HER2v1 (SEQ IDNO: 1). As previously described herein, to enhance the efficacy of thecombination therapy, mutations to the transgenes encoded by therecombinant MVA are made to minimize any potential interaction and/orbinding between the transgene and the administered antibodies.Accordingly, to enhance the efficacy of a therapy involvingco-administration of a HER2 antibody such as, Trastuzumab andPertuzumab, one or more mutations of the HER2 antigen were made. Morespecifically, the relevant antibody binding sites in HER2 were mutated.Thus, in one or more embodiments, the synthetic HER2 protein comprisesone or more mutations in the following amino acid domains: 579-625(Trastuzumab Binding domain), 267-337 (Pertuzumab binding domain),274-288 (HER2 dimerization domain), 721-987 (kinase domain), and1139-1248 (phosphorylation domain). In one or more exemplary embodimentsof the present invention, the HER2 antigen includes one or more of thefollowing mutations: E580A, F595A, K615A, L317A, H318A, D277R, E280K,K753M, Y1023A. Mutations to the HER2v1 antigen (based on HER2(NP_004439.2) are illustrated below.

1 MELAALCRWG LLLALLPPGA ASTQVCTGTD MKLRLPASPE THLDMLRHLY QGCQVVQGNL 61ELTYLPTNAS LSFLQDIQEV QGYVLIAHNQ VRQVPLQRLR IVRGTQLFED NYALAVLDNG 121DPLNNTTPVT GASPGGLREL QLRSLTEILK GGVLIQRNPQ LCYQDTILWK DIFHKNNQLA 181LTLIDTNRSR ACHPCSPMCK GSRCWGESSE DCQSLTRTVC AGGCARCKGP LPTDCCHEQC 241AAGCTGPKHS DCLACLHFNH SGICELHCPA LVTYNTRTFK SMPNPEGRYT FGASCVTACP 301YNYLSTDVGS CTLVCPAANQ EVTAEDGTQR CEKCSKPCAR VCYGLGMEHL REVRAVTSAN 361IQEFAGCKKI FGSLAFLPES FDGDPASNTA PLQPEQLQVF ETLEEITGYL YISAWPDSLP 421DLSVFQNLQV IRGRILHNGA YSLTLQGLGI SWLGLRSLRE LGSGLALIHH NTHLCFVHTV 481PWDQLFRNPH QALLHTANRP EDECVGEGLA CHQLCARGHC WGPGPTQCVN CSQFLRGQEC 541VEECRVLQGL PREYVNARHC LPCHPECQPQ NGSVTCFGPA ADQCVACAHY KDPPACVARC 601PSGVKPDLSY MPIWAFPDEE GACQPCPINC THSCVDLDDK GCPAEQRASP LTSIISAVVG 661ILLVVVLGVV FGILIKRRQQ KIRKYTMARL LQETELVEPL TPSGAMPNQA QMRILKETEL 721RKVKVLGSGA FGTVYKGIWI PDGENVKIPV AIMVLRENTS PKANKEILDE AYVMAGVGSP 781YVSRLLGICL TSTVQLVTQL MPYGCLLDHV RENRGRLGSQ DLLNWCMQIA KGMSYLEDVR 841LVHRDLAARN VLVKSPNHVK ITDFGLARLL DIDETEYHAD GGKVPIKWMA LESILRRAFT 901HQSDVWSYGV TVWELMTFGA KPYDGIPARE IPDLLEKGER LPQPPICTID VYMIMVKCWM 961IDSECRPRFR ELVSEFSRMA RDPQRFVVIQ NEDLGPASPL DSTFYRSLLE DDDMGDLVDA 1021EEALVPQQGF FCPDPAPGAG GMVHHRHASS STRSGGGDLT LGLEPSEEEA PRSPLAPSEG 1081AGSDVFDGDL GMGAAKGLQS LPTHDPSPLQ RYSEDPTVPL PSETDGYVAP LTCSPQPEYV 1141

 

1201

 

LG LDVPV Amino Acid Sequence of HER2 vi. Synthetic HER shown above isbased from HER2 NP_004439.2. Mutated amino acids are shown byunderlines and strikethrough. Amino Acids 579-625 is the binding sitefor Herceptin. Amino Acids 267-337 is the binding site for Perjeta.Amino Acids 274-288 are the residues for the Dimerziation domain.Amino Acids 721-987 are the residues for the kinase domian. AminoAcids 1139-1248 are the residues in the phosphorylation domain. (SEQID NO: 1)

Synthetic HER2 v2

In one or more embodiments, the synthetic HER2 protein is HER2v2 (SEQ IDNO: 13). As previously described herein, to enhance the efficacy of thecombination therapy, mutations to the transgenes encoded by therecombinant MVA are made to minimize any potential interaction and/orbinding between the transgene and the administered antibodies.Accordingly, to enhance the efficacy of a therapy involvingco-administration of a HER2 antibody such as, Trastuzumab andPertuzumab, one or more mutations of the HER2 antigen were made. Morespecifically, the relevant antibody binding sites in HER2 were mutated.Thus, in one or more embodiments, the synthetic HER2 protein comprisesone or more mutations in the following amino acid domains: 579-625(Trastuzumab Binding domain), 267-337 (Pertuzumab binding domain),274-288 (HER2 dimerization domain), 721-987 (kinase domain), and1139-1248 (phosphorylation domain). In one or more exemplary embodimentsof the present invention, the HER2 antigen includes one or more of thefollowing mutations: E580A, F595A, K615A, L317A, H318A, D277R, E280K,K753M, Y1023A.

At least in one aspect, the above mutations enhance the combinationtherapy as they function to minimize any interaction and/or unwantedbinding of MVA-HER2 antigen with the HER2 antibodies Trastuzumab andPertuzumab. Interaction of Trastuzumab with HER2 involves 3 loops in thejuxtamembrane region of HER2 formed by amino acids 579-583 (loop1),592-595 (loop2), as well as 615-625 (loop3) (see, e.g., Cho et al.(2003) Nature 421: 756-60). In those loops there are several keyresidues for HER2-Trastuzumab binding, including E580, D582, P594, F595,K615, Q624S (Satyanarayanajois et al. (2009) Chem. Biol. Drug Des. 74:246-57).

Accordingly, in one embodiment, the HER2 antigen of the presentinvention includes one or more mutations to the residues that interferewith the binding of the HER2 antigen to Trastuzumab. Some exemplaryresidues include, but are not limited to E580, D582, P594, F595, K615,and Q624. Thus, in a more particular embodiment, the HER2 antigen of thepresent invention includes one or more mutations to the residuesselected from the group consisting of E580, D582, P594, F595, K615,Q624, and combination thereof. In yet a more specific embodiment, theHER2 antigen of the present invention includes one or more mutations tothe residues selected from E580, F595 and K615. In still anotherspecific embodiment, the one more mutations to the HER2 antigen includesAla substitutions to E580, F595 and K615 (e.g., E580A, F595A and K615A).

In another exemplary embodiment, the HER2 antigen comprises one or moremutations to the Pertuzumab binding domain. Pertuzumab binds close to aloop in the dimerization domain (domain II) of HER2, involving keyresidues H267, Y274, S310, L317, H318 and K333. Mutations at the bindinginterface strongly reduce binding of Perjeta to HER2 (Franklin et al.(2004) Cancer Cell 5: 317-28). Additional amino acid residues in thisbinding domain potentially contribute to the Pertuzumab-HER2interaction, including F279, V308, and P337.

Accordingly, in one embodiment, the HER2 antigen of the presentinvention includes one or more mutations to the residues that interferewith the binding of the HER2 antigen to Pertuzumab. In a more specificembodiment, the HER2 antigen of the present invention includes one ormore mutations to the residues selected from the group consisting ofH267, F279, V308, S310, L317, H318, K333 and P337, and combinationthereof. In still another specific embodiment, the one or more mutationsto the HER2 antigen includes Ala substitutions to H267, F279, V308,S310, L317, H318, K333 and P337 (e.g. H267A, F279A, V308A, S310A, L317A,H318A, K333A and P337A).

In at least one aspect of the present invention, it was postulated thatif the dimerization of HER2 was minimized this minimizes interaction andbinding between the MVA encoded HER2 and other HER-family members, suchas EGFR, HER3 or HER4, expressed by the MVA-transduced cell. MinimizingHER dimerization would be particularly advantageous as HER2 exerts itsoncogenic potential through intracellular signaling initiated bydimerization. Accordingly, in one embodiment, the HER2 antigen of thepresent invention includes one or more mutations to the residues thatinterfere with dimerization of HER2. Some exemplary residues include,but are not limited to L317, H318, D277, and E280. Thus, in a morespecific embodiment, the HER2 antigen of the present invention includesone or more mutations to the residues selected from the group consistingof L317, H318, D277, E280, and combinations thereof. In a more specificembodiment, the HER2 antigen of the present invention includes one ormore mutations to the residues selected from D277, E280. In stillanother specific embodiment, the one more mutations to the HER2 antigenincludes an Arg substitution to D277 (D277R) and a Lys substitution toE280 (E280K).

In additional aspects, it was postulated that if the tyrosine kinaseactivity was minimized this minimizes downstream activation of cellsignals that might induce cell proliferation and angiogenesis.Accordingly, in one embodiment, the HER2 antigen of the presentinvention includes one or more mutations to the residues that interferewith tyrosine kinase activity of HER2. An exemplary residue includes,but is not limited to K753. Thus, in a more specific embodiment, theHER2 antigen of the present invention includes a mutation to the K753residue. In a more specific embodiment, the HER2 antigen of the presentinvention includes a Met substitution to residue K753 (K753M).

In still additional aspects, it was postulated that if the potentialphosphorylation sites were eliminated this minimizes downstreamactivation of cell signals that might induce cell proliferation andangiogenesis. Accordingly, in one embodiment, the HER2 antigen of thepresent invention includes one or more mutations to the residues thatinterfere with potential phosphorylation sites in HER2. Some exemplaryresidues include, but are not limited to amino acids 1139 to 1248 andY1023. Thus, in a more specific embodiment, the HER2 antigen of thepresent invention includes one or more mutations to the residuesselected from amino acids 1139 to 1248, Y1023, and combination thereof.In a more specific embodiment, the HER2 antigen of the present inventionincludes a deletion of amino acids 1139 to 1248 and/or a substitution ofY1023 to Ala (Y1023A).

In one preferred embodiment, the HER2 antigen comprises one or moremutations to the Pertuzumab binding domain, Trastuzumab binding domain,dimerization domain, kinase domain, and/or phosphorylation domain foundin HER2. Exemplary Mutations to the HER2 antigen (based on NP_004439.2)in the previously described domains are illustrated below as syntheticHER2v2.

1 MELAALCRWG LLLALLPPGA ASTQVCIGTD MKLRLPASPE THLDMLRHLY QGCQVVQGNL 61ELTYLPTNAS LSFLQDIQEV QGYVLIAHNQ VRQVPLQRLR IVRGTQLFED NYALAVLDNG 121DPLNNTTPVT GASPGGLREL QLRSLTEILK GGVLIQRNPQ LCYQDTILWK DIFHYNNQLA 181LTLIDTNRSR ACHPCSPMCK GSRCWGESSE DCQSLTRTVC AGGCARCKGP LPTDCCHEQC 241AAGCTGPKHS DCLACLHFNH SGICELACPA LVTYNTRTAK SMPNPEGRYT FGASCVTACP 301YNYLSTDAGA CTLVCPAANQ EVTAEDGTQR CEACSKACAR VCYGLGMEHL REVRAVTSAN 361IQEFAGCKKI FGSLAFLPES FDGDPASNTA PLQPEQLQVF ETLEEITGYL YISAWPDSLP 421DLSVFQNLQV IRGRILHNGA YSLTLQGLGI SWLGLRSLRE LGSGLALIHH NTHLCFVHTV 481PWDQLFRNPH QALLHTANRP EDECVGEGLA CHQLCARGHC WGPGPTQCVN CSQFLRGQEC 541VEECRVLQGL PREYVNARHC LPCHPECQPQ NGSVTCFGPA ADQCVACAHY KDPPACVARC 601PSGVKPDLSY MPIWAFPDEE GACQPCPINC THSCVDLDDK GCPAEQRASP LTSIISAVVG 661ILLVVVLGVV FGILIKRRQQ KIRKYTMRRL LQETELVEPL TPSGAMPNQA QMRILKETEL 721RKVKVLGSGA FGTVYKGIWI PDGENVKIPV AIMVLRENTS PYANKEILDE AYVMAGVGSP 781YVSRLLGICL TSTVQLVTQL MPYGCLLDHV RENRGRLGSQ DLLNWCMQIA KGMSYLEDVR 841LVHRDLAARN VLVKSPNHVK ITDFGLARLL DIDETEYHAD GGKVPIKWMA LESILRRRFT 901HQSDVWSYGV TVWELMTFGA KPYDGIPARE IPDLLEKGER LPQPPICTID VYMIMVKCWM 961IDSECRPRFR ELVSEFSRMA RDPQRFVVIQ NEDLGPASPL DSTFYRSLLE DDDMGDLVDA 1021EEALVPQQGF FCPDPAPGAG GMVHHRHRSS STRSGGGDLT LGLEPSEEEA PRSPLAPSEG 1091AGSDVMJGDL GMGAAKGLQS LPTHDPSPLQ RYSEDPTVPL PSETDGYVAP LTCSPQPE

1141

1201

 LDVPV Amino Acid Sequence of HER2_v2. (SEQ ID: NO 13) SyntheticHER version2 shown above is based on HER2 NP_004439.2. Mutatedamino acids are shown by underlines and strikethrough. Amino acids 579-625 are the binding site for trastuzumab. Amino acids 267-337 are the binding site for pertuzumab. Amino acids 274-288 are the residuesfor the dimerziation domain. Amino acids 721-987 are the residues forthe kinase domian. Amino acids 1139-1248 are the residues in thephosphorylation domain.

In accordance with the defined advantages herein, in various embodimentsthe present invention includes a synthetic HER2 antigen wherein one ormore amino acids of the HER2 are mutated to prevent the HER2 antigenfrom binding a HER2 antibody. In a more preferred embodiment, thesynthetic HER2 antigen is mutated to prevent binding of an antibodyselected from pertuzumab, trastuzumab, and ado-trastuzumab emtansine.

In other embodiments, the synthetic HER2 antigen comprises one or moremutations to prevent extracellular dimerization, tyrosine kinaseactivity, and/or phosphorylation of the HER2 antigen (once expressed bythe rMVA). In further specific embodiments, the synthetic HER2 antigencomprises one or more mutations to at least one of 3 loops in ajuxtamembrane region of HER2.

In various further embodiments, the present invention includes a nucleicacid encoding a HER2 antigen comprising SEQ ID NO: 1. In additionalembodiments, the present invention includes a nucleic acid encoding aHER2 antigen having at least 90%, 95%, 97% 98%, or 99% identity to SEQID NO:1.

In one embodiment, the present invention includes a nucleic acidcomprising SEQ ID NO:2, which encodes the HER2 antigen of SEQ ID NO:1.In additional embodiments, the present invention includes a nucleic acidencoding a HER2 antigen, the nucleic acid having at least 90%, 95%, 97%98%, or 99% identity to SEQ ID NO:2.

In various additional embodiments, the present invention includes anucleic acid encoding a HER2 antigen comprising SEQ ID NO: 13. Inadditional embodiments, the present invention includes a nucleic acidencoding a HER2 antigen having at least 90%, 95%, 97% 98%, or 99%identity to SEQ ID NO:13.

In one embodiment, the present invention includes a nucleic acidcomprising SEQ ID NO:14, which encodes the HER2 antigen of SEQ ID NO:13.In additional embodiments, the present invention includes a nucleic acidencoding a HER2 antigen, the nucleic acid having at least 90%, 95%, 97%98%, or 99% identity to SEQ ID NO:14.

Synthetic Brachyury Proteins

As illustrated previously, the addition of a second disease/tumorassociate antigen encoded by the MVA enhanced the anti-tumorresponse/activity of the combination therapy. In accordance therewith,in various embodiments of the invention, the recombinant MVAadditionally encodes a Brachyury antigen.

The inventors determined that one or more modifications of the Brachyuryantigens and/or nucleic acids encoding the Brachyury antigens asdescribed herein increase the efficacy of the combination therapy. Morespecifically, one or more mutations were made to the nuclearlocalization signal (NLS) domain in order to minimize and/or avoid anynuclear localization of the Brachyury antigen by a host cell.

Accordingly, in various embodiments, the synthetic Brachyury polypeptideincludes one more mutations to prevent nuclear localization ofBrachyury. “Nuclear localization” can be defined as localization and/ortransport to the nucleus. Conducting assays to determine whether one ormore mutations prevent nuclear localization of a Brachyury antigen iswithin the ordinary skill in the art. Such assays can includeimmunofluorescence analysis and immunofluorescence microscopy.

In more specific embodiments, the synthetic Brachyury polypeptideincludes one more mutations in the NLS domain. In a more specific andpreferred embodiment, the synthetic Brachyury polypeptide has deletedamino acids 286-293 of the Brachyury antigen (GenBank reference NP003172.1), as shown below.

1 MSSPGTESAG KSLQYRVDHL LSAVENELQA GSEKGDPTER ELRVGLEESE LWLRFKELTN 61EMIVTKNGRR MFPVLKVNVS GLDPNAMYSF LLDEVAADNH RWKYVNGEWV PGGKPEPQAP 121SCVYIHPDSP NFGAHWMKAP VSFSKVKLTN KLNGGGQIML NSLHKYEPRI HIVRVGGPQR 181MITSHCFPET QFIAVTAYQN EEITALKIKY NPFAKAFLDA KERSDHKEMM EEPGDSQQPG 241YSQWGWLLPG TSTLCPPANP HPQFGGALSL PSTHSCDRYP 

 

301 PTYSDNSPAC LSMLQSHDNW SSLGMPAHPS MLPVSHNASP PTSSSQYPSL WSVSNGAVTP361 GSQAAAVSNG LGAQFFRGSP AHYTPLTHPV SAPSSSGSPL YEGAAAATDI VDSQYDAAAQ421 GRLIASWTPV SPPSMAmino Acid Sequence of an exemplary modified Brachyury. TheConserved core motif residues RSSPYPSP within a potential NLS ofBrachyury were deleted (shown by strikethrough) (SEQ ID NO:3).

In more specific embodiments, the present invention includes a nucleicacid encoding a Brachyury antigen selected from the group consisting ofSEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO:7. In a preferred aspect, thevarious embodiments of the present invention include a nucleic acidencoding SEQ ID NO:3.

In at least one specific aspect, the nucleic acid encoded by SEQ ID NO:3is particularly advantageous as SEQ ID NO: 3 is configured to preventand/or minimize the Brachyury antigen encoded by the MVA from beinglocalized and/or transported to the nucleus where there may be apossibility of undesired transcriptional activity.

In accordance with defined advantages to the combination therapy, invarious aspects, the embodiments the present invention includes anucleic acid encoding a Brachyury antigen comprising SEQ ID NO: 3. Inadditional aspects, the embodiments of the present invention include anucleic acid encoding a Brachyury antigen having at least 90%, 95%, 97%98%, or 99% identity to SEQ ID NO:3.

In further aspects, the embodiments of the present invention include anucleic acid comprising SEQ ID NO:4, which encodes the Brachyury antigenof SEQ ID NO:3. In additional aspects, the embodiments of the presentinvention include a nucleic acid encoding a Brachyury antigen, thenucleic acid having at least 90%, 95%, 97% 98%, or 99% identity to SEQID NO:4.

CD40L

As illustrated by the present disclosure the inclusion of CD40L as partof the pharmaceutical combination and related method further enhancesthe decrease in tumor volume, prolongs progression-free survival andincrease survival rate realized by the present invention. Thus, invarious embodiments, the pharmaceutical combination further comprisesadministering CD40L to a cancer patient.

While CD40 is constitutively expressed on many cell types, including Bcells, macrophages, and dendritic cells, its ligand CD40L ispredominantly expressed on activated T helper cells. The cognateinteraction between dendritic cells and T helper cells early afterinfection or immunization ‘licenses’ dendritic cells to prime CTLresponses. Dendritic cell licensing results in the up-regulation ofco-stimulatory molecules, increased survival and better cross-presentingcapabilities. This process is mainly mediated via CD40/CD40Linteraction. However, various configurations of CD40L are described,from membrane bound to soluble (monomeric to trimeric) which inducediverse stimuli, either inducing or repressing activation,proliferation, and differentiation of APCs.

As shown by the results of Example 19, the MVA encoded CD40L presents amuch safer alternative to a cancer patient, as the CD40L encoded by MVApresents decreased toxicity compared to as a soluble CD40 agonist.

In one or more preferred embodiments, CD40L is encoded by the MVA of thepresent invention. In even more preferred embodiments, the CD40Lcomprises a nucleic acid encoding SEQ ID NO: 11. In still more preferredembodiments, the CD40L comprises a nucleic acid having at least 90%,95%, 97% 98%, or 99% identity to SEQ ID NO:12. In a most preferredembodiment, the CD40L comprises SEQ ID NO:12.

Exemplary Antibodies

In various embodiments, the pharmaceutical combination and relatedmethods include an antibody specific to an antigen that is expressed onthe cell membrane of a tumor cell. It is understood in the art that inmany cancers, one or more antigens are expressed or overexpressed on thetumor cell membrane (see, e.g., Duerig et al. (2002) Leukemia 16: 30-5;Mocellin et al. (2013) Biochim. Biophys. Acta 1836: 187-96; Arteaga(2017); Finn (2018) J. Immunol. 200: 385-91; Ginaldi et al. (1998) J.Clin. Pathol. 51: 364-9). Assays for determining whether an antigen isexpressed or overexpressed on a tumor cells are readily understood inthe art (Id), as well as methods for producing antibodies to aparticular antigen.

In more specific embodiments, the pharmaceutical combination and relatedmethods include an antibody, wherein in the antibody is a) specific toan antigen that is expressed on a cell membrane of a tumor and b)comprises an Fc domain. In at least one aspect, the characteristics ofthe antibody (e.g., a) and b)) enable the antibody to bind to andinteract with an effector cell, such as an NK cell, macrophage,basophil, neutrophil, eosinophil, monocytes, mast cells, and/ordendritic cells, and enable the antibody to bind a tumor antigen that isexpressed on a tumor cell. In a preferred embodiment, the antibodycomprises an Fc domain. In an additional preferred embodiment, theantibody is able to bind and interact with an NK cell.

Some exemplary antibodies to antigens expressed on tumor cells that are30 contemplated by the present disclosure include, but are not limitedto, anti-CD20 (e.g., rituximab, ofatumumab, tositumomab), anti-CD52(e.g., alemtuzumab, Campath® antibody), anti-EGFR (e.g., cetuximab,Erbitux® antibody, panitumumab), anti-CD2 (e.g., siplizumab), anti-CD37(e.g., BI836826), anti-CD123 (e.g., JNJ-56022473), anti-CD30 (e.g.,XmAb2513), anti-CD38 (e.g., daratumumab, Darzalex® antibody), anti-PDL1(e.g., avelumab, atezolilzumab, durvalumab), anti-GD2 (e.g., 3F8,ch14.18, KW-2871, dinutuximab), anti-CEA, anti-MUC1, anti-FLT3,anti-CD19, anti-CD40, anti-SLAMF7, anti-CCR4, anti-B7-H3, anti-ICAM1,anti-CSF1R, anti-CA125 (e.g., oregovomab), anti-FRα (e.g. MOv18-IgG1,mirvetuximab soravtansine (IMGN853), MORAb-202), anti-mesothelin (e.g.MORAb-009) and anti-HER2 (e.g., trastuzumab, Herzuma® antibody, ABP 980,and/or pertuzumab).

In a more preferred embodiment, the antibody included as part of presentinvention includes an antibody that when administered to a patient bindsto the corresponding antigen on a tumor cell and induces antibodydependent cell-mediated cytotoxicity (ADCC). In an even more preferredembodiment, the antibody comprises an antibody that is approved or inpre-approval for the treatment of a cancer.

In even more preferred embodiments, the antibody is an anti-HER2antibody. In a most preferred embodiment, antibody is selected frompertuzumab, trastuzumab, Herzuma® antibody, ABP 980, and ado-trastuzumabemtansine.

In additional embodiments, the antibody comprises a fusion of one ormore antibodies and/or antibody fragments. Exemplary fusion antibodiesand/or antibody fragments include, but are not limited to BispecificKiller cell Engagers (BiKE) and Trispecific Killer cell Engagers(TriKE). BiKEs and TriKEs are known to effectively drive NK cellanti-tumor effects and enable NK cell-mediated ADCC (see, e.g., Tay etal. (2016) Hum. Vaccin. Immunother. 12: 2790-96). It is contemplatedthat the 161533 TriKE and/or the 1633 BiKE can be used as the antibodyin the present invention. It is additionally contemplated that theantibodies of the present invention can be embodied as Bispecific T cellengagers, or BiTEs (see, e.g., Huehls et al. (2015) Immunol. Cell Biol.93: 290-296).

Recombinant Poxviruses

In one or more aspects of the present invention, the nucleotides andproteins sequences of the present disclosure can be included in arecombinant poxvirus.

In the various embodiments of the present disclosure, the recombinantpoxvirus is preferably an orthopoxvirus such as, but not limited to, avaccinia virus, a Modified Vaccinia Ankara (MVA) virus, MVA-BN, or aderivative of MVA-BN.

Examples of vaccinia virus strains are the strains Temple of Heaven,Copenhagen, Paris, Budapest, Dairen, Gam, MRIVP, Per, Tashkent, TBK,Tom, Bern, Patwadangar, BIEM, B-15, Lister, EM-63, New York City Boardof Health, Elstree, Ikeda and WR. A preferred vaccinia virus (VV) strainis the Wyeth (DRYVAX) strain (U.S. Pat. No. 7,410,644).

Recombinant MVA

In more preferred embodiments of the present invention, the one or moreproteins and nucleotides disclosed herein are included in a recombinantMVA. As described and illustrated by the present disclosure, theintravenous administration of the recombinant MVAs of the presentdisclosure induces in various aspects an enhanced immune response incancer patients. Thus, in one or more preferred embodiments, theinvention includes a recombinant MVA comprising one or more nucleicacids encoding HER2, Brachyury, and/or CD40L described herein. In morepreferred embodiments, the recombinant MVA comprises one or more nucleicacids encoding the synthetic HER2 and synthetic Brachyury antigensdescribed herein. In another more preferred embodiment, the recombinantMVA comprises one or more nucleic acids encoding the synthetic HER2 andthe synthetic Brachyury antigens described herein as well as CD40L. Instill another more preferred embodiment, the recombinant MVA comprisesone or more nucleic acids encoding SEQ ID NO:1 or SEQ ID NO: 13 (Her2),and SEQ ID NO: 3 (Brachyury). In yet another preferred embodiment, therecombinant MVA comprises one or more nucleic acids encoding SEQ ID NO:1or SEQ ID NO: 13, SEQ ID NO: 3, and CD40L. In a more preferredembodiment, the recombinant MVA comprises one or more nucleic acidsencoding SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 11, and/or SEQ ID NO: 13.In a most preferred embodiment, the recombinant MVA comprises one ormore nucleic acids encoding SEQ ID NO:13, SEQ ID NO:3, and SEQ ID NO:11

In additional embodiments, the recombinant MVA comprises one or morenucleic acids selected from the group consisting of SEQ ID NO:2, SEQ IDNO: 4, and/or SEQ ID NO: 14. In another more preferred embodiment, therecombinant MVA comprises one or more nucleic acids selected from thegroup consisting of SEQ ID NO:2, SEQ ID NO:4, and CD40L. In stillanother preferred embodiment, the recombinant MVA comprises one or morenucleic acids selected from the group consisting of SEQ ID NO:2, SEQ IDNO: 4, and SEQ ID NO: 12. In a more preferred embodiment, therecombinant MVA comprises SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 12.In a most preferred embodiment, the recombinant MVA comprises SEQ ID NO:14, SEQ ID NO: 4, and SEQ ID NO:12.

Example of MVA virus strains that are useful in the practice of thepresent invention and that have been deposited in compliance with therequirements of the Budapest Treaty are strains MVA 572, deposited atthe European Collection of Animal Cell Cultures (ECACC), VaccineResearch and Production Laboratory, Public Health Laboratory Service,Centre for Applied Microbiology and Research, Porton Down, Salisbury,Wiltshire SP4 0JG, United Kingdom, with the deposition number ECACC94012707 on Jan. 27, 1994, and MVA 575, deposited under ECACC 00120707on Dec. 7, 2000, MVA-BN, deposited on Aug. 30, 2000 at the EuropeanCollection of Cell Cultures (ECACC) under number V00083008, and itsderivatives, are additional exemplary strains.

“Derivatives” of MVA-BN refer to viruses exhibiting essentially the samereplication characteristics as MVA-BN, as described herein, butexhibiting differences in one or more parts of their genomes. MVA-BN, aswell as derivatives thereof, are replication incompetent, meaning afailure to reproductively replicate in vivo and in vitro. Morespecifically in vitro, MVA-BN or derivatives thereof have been describedas being capable of reproductive replication in chicken embryofibroblasts (CEF), but not capable of reproductive replication in thehuman keratinocyte cell line HaCat (Boukamp et al. (1988), J. Cell Biol.106: 761-771), the human bone osteosarcoma cell line 143B (ECACC DepositNo. 91112502), the human embryo kidney cell line 293 (ECACC Deposit No.85120602), and the human cervix adenocarcinoma cell line HeLa (ATCCDeposit No. CCL-2). Additionally, MVA-BN or derivatives thereof have avirus amplification ratio at least two-fold less, more preferablythree-fold less than MVA-575 in Hela cells and HaCaT cell lines. Testsand assay for these properties of MVA-BN and derivatives thereof aredescribed in WO 02/42480 (issued as U.S. Pat. No. 6,913,752) and WO03/048184 (U.S. Pat. No. 7,759,116).

The term “not capable of reproductive replication” or “no capability ofreproductive replication” in human cell lines in vitro as described inthe previous paragraphs is, for example, described in WO 02/42480, whichalso teaches how to obtain MVA having the desired properties asmentioned above. The term applies to a virus that has a virusamplification ratio in vitro at 4 days after infection of less than 1using the assays described in WO 02/42480 or in U.S. Pat. No. 6,761,893.

The term “failure to reproductively replicate” refers to a virus thathas a virus amplification ratio in human cell lines in vitro asdescribed in the previous paragraphs at 4 days after infection of lessthan 1. Assays described in WO 02/42480 or in U.S. Pat. No. 6,761,893are applicable for the determination of the virus amplification ratio.

The amplification or replication of a virus in human cell lines in vitroas described in the previous paragraphs is normally expressed as theratio of virus produced from an infected cell (output) to the amountoriginally used to infect the cell in the first place (input) referredto as the “amplification ratio.” An amplification ratio of “1” definesan amplification status where the amount of virus produced from theinfected cells is the same as the amount initially used to infect thecells, meaning that the infected cells are permissive for virusinfection and reproduction. In contrast, an amplification ratio of lessthan 1, i.e., a decrease in output compared to the input level,indicates a lack of reproductive replication and therefore attenuationof the virus.

In another embodiment, the recombinant poxvirus, including the syntheticnucleotides and proteins of the present invention can be embodied in anavipoxvirus, such as but not limited to, a fowlpox virus.

The term “avipoxvirus” refers to any avipoxvirus, such as Fowlpoxvirus,Canarypoxvirus, Uncopoxvirus, Mynahpoxvirus, Pigeonpoxvirus,Psittacinepoxvirus, Quailpoxvirus, Peacockpoxvirus, Penguinpoxvirus,Sparrowpoxvirus, Starlingpoxvirus and Turkeypoxvirus. Preferredavipoxviruses are Canarypoxvirus and Fowlpoxvirus.

An example of a canarypox virus is strain Rentschler. A plaque purifiedCanarypox strain termed ALVAC (U.S. Pat. No. 5,766,598) was depositedunder the terms of the Budapest treaty with the American Type CultureCollection (ATCC), accession number VR-2547. Another Canarypox strain isthe commercial canarypox vaccine strain designated LF2 CEP 524 24 10 75,available from Institute Merieux, Inc.

Examples of a Fowlpox virus are strains FP-1, FP-5, TROVAC (U.S. Pat.No. 5,766,598), POXVAC-TC (U.S. Pat. No. 7,410,644), TBC-FPV (TherionBiologics-FPV), FP-1 is a Duvette strain modified to be used as avaccine in one-day old chickens. The strain is a commercial fowlpoxvirus vaccine strain designated O DCEP 25/CEP67/239 Oct. 1980 and isavailable from Institute Merieux, Inc. FP-5 is a commercial fowlpoxvirus vaccine strain of chicken embryo origin available from AmericanScientific Laboratories (Division of Schering Corp., Madison, Wis.,United States Veterinary License No. 165, serial No. 30321).

Expression Cassettes/Control Sequences

In various aspects, the one or more nucleic acids described herein areembodied in in one or more expression cassettes in which the one or morenucleic acids are operatively linked to expression control sequences.“Operably linked” means that the components described are inrelationship permitting them to function in their intended manner e.g.,a promoter to transcribe the nucleic acid to be expressed. An expressioncontrol sequence operatively linked to a coding sequence is joined suchthat expression of the coding sequence is achieved under conditionscompatible with the expression control sequences. The expression controlsequences include, but are not limited to, appropriate promoters,enhancers, transcription terminators, a start codon at the beginning aprotein-encoding open reading frame, splicing signals for introns, andin-frame stop codons. Suitable promoters include, but are not limitedto, the SV40 early promoter, an RSV promoter, the retrovirus LTR, theadenovirus major late promoter, the human CMV immediate early Ipromoter, and various poxvirus promoters including, but not limited tothe following vaccinia virus or MVA-derived and FPV-derived promoters:the 30K promoter, the 13 promoter, the PrS promoter, the PrS5E promoter,the Pr7.5K, the PrHyb promoter, the Pr13.5 long promoter, the 40Kpromoter, the MVA-40K promoter, the FPV 40K promoter, 30k promoter, thePrSynIIm promoter, the PrLE1 promoter, and the PR1238 promoter.Additional promoters are further described in WO 2010/060632, WO2010/102822, WO 2013/189611, WO 2014/063832, and WO 2017/021776 whichare incorporated fully by reference herein.

Additional expression control sequences include, but are not limited to,leader sequences, termination codons, polyadenylation signals and anyother sequences necessary for the appropriate transcription andsubsequent translation of the nucleic acid sequence encoding the desiredrecombinant protein (e.g., HER2, Brachyury, and/or CD40L) in the desiredhost system. The poxvirus vector may also contain additional elementsnecessary for the transfer and subsequent replication of the expressionvector containing the nucleic acid sequence in the desired host system.It will further be understood by one skilled in the art that suchvectors are easily constructed using conventional methods (Ausubel etal. (1987) in “Current Protocols in Molecular Biology,” John Wiley andSons, New York, N.Y.) and are commercially available.

In certain embodiments, the one or more recombinant MVAs of the presentdisclosure comprises one or more cytokines, such as IL-2, IL-6, IL-12,IL-15, IL-7, IL-21 RANTES, GM-CSF, TNF-α, or IFN-γ, one or more growthfactors, such as GM-CSF or G-CSF, one or more costimulatory molecules,such as ICAM-1, LFA-3, CD72, B7-1, B7-2, or other B7 related molecules;one or more molecules such as CD70, OX-40L or 4-1 BBL, or combinationsof these molecules, can be used as biological adjuvants (see, forexample, Salgaller et al. (1998) J. Surg. Oncol. 68(2):122-38; Lotze etal. (2000) Cancer i Sci. Am. 6 (Suppl. 1): S61-6; Cao et al. (1998) StemCells 16 (Suppl 1): 251-60; Kuiper et al. (2000) Adv. Exp. Med. Biol.465: 381-90). These molecules can be administered systemically (orlocally) to the host. In several examples, IL-2, RANTES, GM-CSF, TNF-α,IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, B7-1 B7-2, CD70, OX-40L, 4-1 BBLand ICAM-1 are administered.

Methods and Dosing Regimens for Administering the PharmaceuticalCombination

In still further aspects, the present disclosure provides for one ormore regimens for administration of the pharmaceutical combinationand/or methods of the present invention. In at least one aspect, theregimens of the present invention increase the effectiveness of thepharmaceutical combination and/or therapy to reduce tumor volume andincrease survival rate of a cancer patient.

Thus, in one embodiment, there is a pharmaceutical combination and/ormethod for reducing tumor size and/or increasing survival in a cancerpatient comprising administering to the cancer patient a pharmaceuticalcombination of the present disclosure, wherein the recombinant MVA isadministered at the same time or after administration of the antibody.

In one specific exemplary embodiment, there is a pharmaceuticalcombination and/or method for reducing tumor size and/or increasingsurvival in a cancer patient comprising a) administering to the cancerpatient an antibody, wherein the antibody comprises an Fc domain and isspecific to an antigen that is expressed on the cell membrane of a tumorcell; and b) intravenously administering to the cancer patient arecombinant modified vaccinia virus Ankara (MVA) comprising a firstnucleic acid encoding a first heterologous tumor-associated antigen(TAA) that when administered intravenously induces both an enhancedNatural Killer (NK) cell response and an enhanced cytotoxic T cellresponse; wherein b) is administered at the same time or after a).

In several specific embodiments, the recombinant MVA can be administeredat the same time as the antibody. In other specific embodiments, therecombinant MVA is administered after or subsequent to administration ofthe antibody. In various embodiments, the recombinant MVA isadministered within 1 to 6 days after the antibody. In more preferredembodiments, the recombinant MVA is administered within 0 to 5 days, 0to 4 days, 0 to 3 days, or 0-2 days after administration of theantibody. In most preferred embodiment, the recombinant MVA isadministered 0 to 3 days after the antibody is administered.

In various additional embodiments, the recombinant MVA is administeredwithin 1 to 6 days after the antibody. In more preferred embodiments,the recombinant MVA is administered within 1 to 5 days, 1 to 4 days, 1to 3 days, or 1-2 days after administration of the antibody. In mostpreferred embodiment, the recombinant MVA is administered 1 to 3 daysafter the antibody is administered.

In one advantageous aspect of the present invention, administering therecombinant MVA at the same time or after the antibody enables theadministered antibody to bind tumor cells at the same time or prior toenhancement of the NK cell response that results from administering therecombinant MVA. Accordingly, in an exemplary first step, an antibody isadministered resulting in the antibody binding to the antigen on thesurface of one or more tumor cells. In an exemplary second step, arecombinant MVA is intravenously administered which as described herein,enhances and/or increases both the subject's innate immune response(e.g., the NK cell response) and T cell response. The enhanced innateimmune response aggressively targets and kills tumor cells having thebound antibody.

In another advantageous aspect, the regimens of the present inventionare designed to effectively attack the tumor with the both the subject'sinnate immune and adaptive immune responses. In one exemplary aspect,the pharmaceutical combination is designed to a recombinant MVAintravenously administered at the same time or after the antibodyadministration, the recombinant MVA inducing an enhanced NK and innateimmune response that attacks and kill those tumor cells having boundantibody. During the period in which the innate immune response iskilling antibody-coated tumor cells, the recombinant MVA is alsoinducing a specific T cell response (including both CD8 and CD4 T cells)in the patient. The disclosed regimen is designed such that as theinnate immune response e.g., NK cells, is naturally declining, anenhanced specific CD8/CD4 T cell response is killing tumor cells. Thedesigned regimen is particularly advantageous, as the enhanced specificCD8/CD4 T cell response can kill those tumor cells that have potentiallyevaded a patient's innate and/or non-specific immune response.

In other aspects, the pharmaceutical combination of the presentinvention is administered as part of a homologous and/or heterologousprime-boost regimen. Illustrated in FIGS. 8-13, a homologous and/orheterologous prime boost regimen prolongs and reactivates enhanced NKcell responses as well as increases a subject's specific CD8 and CD4 Tcell responses. Thus, in one or more embodiments there is apharmaceutical combination and/or method for a reducing tumor sizeand/or increasing survival in a cancer patient comprising administeringto the cancer patient a pharmaceutical combination of the presentdisclosure, wherein the pharmaceutical combination is administered aspart of a homologous or heterologous prime-boost regimen. In morepreferred aspect of the prime-boost regimen, the recombinant MVA isintravenously administered at the same time or after to theadministration of the antibody.

Generation of Recombinant MVA Viruses Comprising Transgenes

The recombinant MVA viruses provided herein can be generated by routinemethods known in the art. Methods to obtain recombinant poxviruses or toinsert exogenous coding sequences into a poxviral genome are well knownto the person skilled in the art. For example, methods for standardmolecular biology techniques such as cloning of DNA, DNA and RNAisolation, Western blot analysis, RT-PCR and PCR amplificationtechniques are described in Molecular Cloning, A Laboratory Manual (2nded., Sambrook et al., Cold Spring Harbor Laboratory Press (1989)), andtechniques for the handling and manipulation of viruses are described inVirology Methods Manual (Mahy et al. (eds.), Academic Press (1996)).Similarly, techniques and know-how for the handling, manipulation andgenetic engineering of MVA are described in Molecular Virology: APractical Approach (Davison & Elliott (eds.), The Practical ApproachSeries, IRL Press at Oxford University Press, Oxford, U K (1993); see,e.g., “Chapter 9: Expression of genes by Vaccinia virus vectors”) andCurrent Protocols in Molecular Biology (John Wiley & Son, Inc. (1998);see, e.g., “Chapter 16, Section IV: Expression of proteins in mammaliancells using vaccinia viral vector”).

For the generation of the various recombinant MVA viruses disclosedherein, different methods may be applicable. The DNA sequence to beinserted into the virus can be placed into an E. coli plasmid constructinto which DNA homologous to a section of DNA of the poxvirus has beeninserted. Separately, the DNA sequence to be inserted can be ligated toa promoter. The promoter-gene linkage can be positioned in the plasmidconstruct so that the promoter-gene linkage is flanked on both ends byDNA homologous to a DNA sequence flanking a region of poxviral DNAcontaining a non-essential locus. The resulting plasmid construct can beamplified by propagation within E. coli bacteria and isolated. Theisolated plasmid containing the DNA gene sequence to be inserted can betransfected into a cell culture, e.g., of chicken embryo fibroblasts(CEFs), at the same time the culture is infected with MVA virus.Recombination between homologous MVA viral DNA in the plasmid and theviral genome, respectively, can generate a poxvirus modified by thepresence of foreign DNA sequences.

According to a preferred embodiment, a cell of a suitable cell cultureas, e.g., CEF cells, can be infected with a MVA virus. The infected cellcan be, subsequently, transfected with a first plasmid vector comprisinga foreign or heterologous gene or genes, such as one or more of thenucleic acids provided in the present disclosure; preferably under thetranscriptional control of a poxvirus expression control element. Asexplained above, the plasmid vector also comprises sequences capable ofdirecting the insertion of the exogenous sequence into a selected partof the MVA viral genome. Optionally, the plasmid vector also contains acassette comprising a marker and/or selection gene operably linked to apoxviral promoter. Suitable marker or selection genes are, e.g., thegenes encoding the green fluorescent protein, β-galactosidase,neomycin-phosphoribosyltransferase or other markers. The use ofselection or marker cassettes simplifies the identification andisolation of the generated recombinant poxvirus. However, a recombinantpoxvirus can also be identified by PCR technology. Subsequently, afurther cell can be infected with the recombinant poxvirus obtained asdescribed above and transfected with a second vector comprising a secondforeign or heterologous gene or genes. In case, this gene shall beintroduced into a different insertion site of the poxviral genome, thesecond vector also differs in the poxvirus-homologous sequencesdirecting the integration of the second foreign gene or genes into thegenome of the poxvirus. After homologous recombination has occurred, therecombinant virus comprising two or more foreign or heterologous genescan be isolated. For introducing additional foreign genes into therecombinant virus, the steps of infection and transfection can berepeated by using the recombinant virus isolated in previous steps forinfection and by using a further vector comprising a further foreigngene or genes for transfection.

Alternatively, the steps of infection and transfection as describedabove are interchangeable, i.e., a suitable cell can at first betransfected by the plasmid vector comprising the foreign gene and, then,infected with the poxvirus. As a further alternative, it is alsopossible to introduce each foreign gene into different viruses,co-infect a cell with all the obtained recombinant viruses and screenfor a recombinant including all foreign genes. A third alternative isligation of DNA genome and foreign sequences in vitro and reconstitutionof the recombined vaccinia virus DNA genome using a helper virus. Afourth alternative is homologous recombination in E. coli or anotherbacterial species between a MVA virus genome cloned as a bacterialartificial chromosome (BAC) and a linear foreign sequence flanked withDNA sequences homologous to sequences flanking the desired site ofintegration in the MVA virus genome.

The one or more nucleic acids of the present disclosure may be insertedinto any suitable part of the MVA virus or MVA viral vector. Suitableparts of the MVA virus are non-essential parts of the MVA genome.Non-essential parts of the MVA genome may be intergenic regions or theknown deletion sites 1-6 of the MVA genome. Alternatively oradditionally, non-essential parts of the recombinant MVA can be a codingregion of the MVA genome which is non-essential for viral growth.However, the insertion sites are not restricted to these preferredinsertion sites in the MVA genome, since it is within the scope of thepresent invention that the nucleic acids of the present invention (e.g.,HER2, Brachyury, and CD40L) and any accompanying promoters as describedherein may be inserted anywhere in the viral genome as long as it ispossible to obtain recombinants that can be amplified and propagated inat least one cell culture system, such as Chicken Embryo Fibroblasts(CEF cells).

Preferably, the nucleic acids of the present invention may be insertedinto one or more intergenic regions (IGR) of the MVA virus. The term“intergenic region” refers preferably to those parts of the viral genomelocated between two adjacent open reading frames (ORF) of the MVA virusgenome, preferably between two essential ORFs of the MVA virus genome.For MVA, in certain embodiments, the IGR is selected from IGR 07/08, IGR44/45, IGR 64/65, IGR 88/89, IGR 136/137, and IGR 148/149.

For MVA virus, the nucleotide sequences may, additionally oralternatively, be inserted into one or more of the known deletion sites,i.e., deletion sites I, II, III, IV, V, or VI of the MVA genome. Theterm “known deletion site” refers to those parts of the MVA genome thatwere deleted through continuous passaging on CEF cells characterized at20 passage 516 with respect to the genome of the parental virus fromwhich the MVA is derived from, in particular the parentalchorioallantois vaccinia virus Ankara (CVA) e.g., as described inMeisinger-Henschel et al. ((2007) J. Gen. Virol. 88: 3249-59).

Vaccines

In certain embodiments, the recombinant MVA of the present disclosurecan be formulated as part of a vaccine. For the preparation of vaccines,the MVA virus can be converted into a physiologically acceptable form.In certain embodiments, such preparation is based on experience in thepreparation of poxvirus vaccines used for vaccination against smallpox,as described, for example, in Stickl et al. ((1974) Dtsch. med. Wschr.99: 2386-2392)

An exemplary preparation follows. Purified virus is stored at −80° C.with a titer of 5×10⁸ TCID50/ml formulated in 10 mM Tris, 140 mM NaCl,pH 7.4. For the preparation of vaccine shots, e.g., 1×10⁸−1×10⁹particles of the virus can be lyophilized in phosphate-buffered saline(PBS) in the presence of 2% peptone and 1% human albumin in an ampoule,preferably a glass ampoule. Alternatively, the vaccine shots can beprepared by stepwise, freeze-drying of the virus in a formulation. Incertain embodiments, the formulation contains additional additives suchas mannitol, dextran, sugar, glycine, lactose, polyvinylpyrrolidone, orother additives, such as, including, but not limited to, antioxidants orinert gas, stabilizers or recombinant proteins (e.g. human serumalbumin) suitable for in vivo administration. The ampoule is then sealedand can be stored at a suitable temperature, for example, between 4° C.and room temperature for several months. However, as long as no needexists, the ampoule is stored preferably at temperatures below −20° C.,most preferably at about −80° C.

In various embodiments involving vaccination or therapy, thelyophilisate is dissolved in 0.1 to 0.5 ml of an aqueous solution,preferably physiological saline or Tris buffer such as 10 mM Tris, 140mM NaCl pH 7.7. It is contemplated that the recombinant MVA, vaccine orpharmaceutical composition of the present disclosure can be formulatedin solution in a concentration range of 10⁴ to 10¹⁰ TCID₅₀/ml, 105 to5×10⁸ TCID₅₀/ml, 10⁶ to 10⁸ TCID₅₀/ml, or 10⁷ to 10⁸ TCID₅₀/ml. Apreferred dose for humans comprises between 10⁶ to 10¹⁰ TCID₅₀,including a dose of 10⁶ TCID₅₀, 10⁷ TCID₅₀, 10⁸ TCID₅₀, 5×10′TCID₅₀, 109TCID₅₀, 5×10⁹ TCID₅₀, or 10¹⁰ TCID₅₀. Optimization of dose and number ofadministrations is within the skill and knowledge of one skilled in theart.

In one or more preferred embodiments, as set forth herein, therecombinant MVA is administered to a cancer patient intravenously.

In additional embodiments, the antibody can be administered eithersystemically or locally, i.e., by intraperitoneal, parenteral,subcutaneous, intravenous, intramuscular, intranasal, intradermal, orany other path of administration known to a skilled practitioner.

Kits, Compositions, and Methods of Use

In various embodiments, the invention encompasses kits, pharmaceuticalcombinations, pharmaceutical compositions, and/or immunogeniccombination, comprising the a) recombinant MVA that includes the nucleicacids described herein and b) one or more antibodies described herein.

It is contemplated that the kit and/or composition can comprise one ormultiple containers or vials of a recombinant poxvirus of the presentdisclosure, one or more containers or vials of an antibody of thepresent disclosure, together with instructions for the administration ofthe recombinant MVA and antibody. It is contemplated that in a moreparticular embodiment, the kit can include instructions foradministering the recombinant MVA and antibody in a first primingadministration and then administering one or more subsequent boostingadministrations of the recombinant MVA and antibody.

The kits and/or compositions provided herein may generally include oneor more pharmaceutically acceptable and/or approved carriers, additives,antibiotics, preservatives, adjuvants, diluents and/or stabilizers. Suchauxiliary substances can be water, saline, glycerol, ethanol, wetting oremulsifying agents, pH buffering substances, or the like. Suitablecarriers are typically large, slowly metabolized molecules such asproteins, polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers, lipid aggregates, or thelike.

Certain Exemplary Embodiments

Embodiment 1 is a method of reducing tumor size and/or increasingsurvival in a cancer patient, the method comprising: a) intravenouslyadministering to the cancer patient a recombinant modified vacciniavirus Ankara (MVA) comprising a first nucleic acid encoding a firstheterologous tumor-associated antigen (TAA) that when administeredintravenously induces both an enhanced Natural Killer (NK) cell responseand an enhanced cytotoxic T cell response as compared to an NK cellresponse and a T cell response induced by a non-intravenousadministration of a recombinant MVA comprising a first nucleic acidencoding a first heterologous cancer-associated antigen; and b)administering to the cancer patient an antibody, wherein the antibodycomprises an Fc domain and is specific to an antigen that is expressedor over-expressed on the cell membrane of a tumor cell; whereinadministration of a) and b) to the cancer patient reduces tumor size inthe cancer patient and/or increases the survival rate of the cancerpatient as compared to a non-intravenous administration of a) or anadministration of b) alone.

Embodiment 2 is a method of reducing tumor size and/or increasingsurvival in a cancer patient, the method comprising: (a) administeringto the cancer patient an antibody, wherein the antibody comprises an Fcdomain and is specific to an antigen that is expressed on the cellmembrane of a tumor cell; and (b) intravenously administering to thecancer patient a recombinant modified vaccinia virus Ankara (MVA)comprising a first nucleic acid encoding a first heterologoustumor-associated antigen (TAA) that when administered intravenouslyinduces both an enhanced Natural Killer (NK) cell response and anenhanced cytotoxic T cell response as compared to an NK cell responseand a T cell response induced by a non-intravenous administration of arecombinant MVA comprising a first nucleic acid encoding a firstheterologous cancer-associated antigen; wherein administration of (a)and (b) to the cancer patient reduces tumor size in the cancer patientand/or increases the survival rate of the cancer patient as compared toan non-intravenous administration of (a) or an administration of (b)alone.

Embodiment 3 is a method of reducing tumor size and/or increasingsurvival in a cancer patient, the method comprising: (a) administeringto the cancer patient an antibody, wherein the antibody comprises an Fcdomain and is specific to an antigen that is expressed on the outersurface of cell membrane of a tumor cell that when administered inducesADCC in the cancer patient; and (b) intravenously administering to thecancer patient a recombinant modified vaccinia virus Ankara (MVA)comprising a first nucleic acid encoding a first heterologoustumor-associated antigen (TAA) that when administered intravenouslyinduces both an enhanced Natural Killer (NK) cell response and anenhanced T cell response as compared to an NK cell response and a T cellresponse induced by a non-intravenous administration of a recombinantMVA comprising a first nucleic acid encoding a first heterologouscancer-associated antigen; wherein administration of (a) and (b) to thecancer patient reduces tumor size in the cancer patient and/or increasesthe survival rate of the cancer patient as compared to annon-intravenous administration of (a) or an administration of (b) alone.

Embodiment 4 is the method of any one of embodiments 1-3, wherein theantibody is specific to an antigen that is overexpressed on the cellmembrane of the tumor cell.

Embodiment 5 is the method of any one of Embodiments 1-4 furthercomprising intravenously administering CD40L to the cancer patient.

Embodiment 6 is the method of any one of Embodiments 1-5, wherein theCD40L is encoded by the MVA.

Embodiment 7 is the method of any one of Embodiments 1-6, whereininducing an enhanced NK cell response comprises at least one of a)inducing an enhanced ADCC response and b) inducing NK cells to targetand kill tumor cells having low MHC expression.

Embodiment 8 is the method of any one of Embodiments 1-6, whereininducing an enhanced NK cell response comprises inducing an enhancedADCC response.

Embodiment 9 is the method of any one of Embodiments 1-8, wherein therecombinant MVA is administered at the same time or after the antibody.

Embodiment 10 is the method of any one of Embodiments 1-8, wherein therecombinant MVA is administered after the antibody.

Embodiment 11 is the method of any one of Embodiments 1-10, wherein thethe MVA further comprises a second nucleic acid encoding a secondheterologous TAA.

Embodiment 12 is the method of any one of Embodiments 1-11, wherein thefirst and/or second TAA is selected from the group consisting of:5-α-reductase, α-fetoprotein (“AFP”), AM-1, APC, April, B melanomaantigen gene (“BAGE”), β-catenin, Bcl12, bcr-abl, Brachyury, CA-125,caspase-8 (“CASP-8”, also known as “FLICE”), Cathepsins, CD19, CD20,CD21/complement receptor 2 (“CR2”), CD22/BL-CAM, CD23/FcsRII, CD33,CD35/complement receptor 1 (“CR1”), CD44/PGP-1, CD45/leucocyte commonantigen (“LCA”), CD46/membrane cofactor protein (“MCP”), CD52/CAMPATH-1,CD55/decay accelerating factor (“DAF”), CD59/protectin, CDCl27, CDK4,carcinoembryonic antigen (“CEA”), c-myc, cyclooxygenase-2 (“cox-2”),deleted in colorectal cancer gene (“DCC”), DcR3, E6/E7, CGFR, EMBP,Dna78, farnesyl transferase, fibroblast growth factor-8a (“FGF8a”),fibroblast growth factor-8b (“FGF8b”), FLK-1/KDR, folic acid receptor,G250, G melanoma antigen gene family (“GAGE-family”), gastrin 17,gastrin-releasing hormone, ganglioside 2 (“GD2”)/ganglioside 3(“GD3”)/ganglioside-monosialic acid-2 (“GM2”), gonadotropin releasinghormone (“GnRH”), UDP-GlcNAc:R₁Man(α1-6)R₂ [GlcNAc to Man(α1-6)]β1,6-N-acetylglucosaminyltransferase V (“GnT V”), GP1, gp100/Pme 117,gp-100-in4, gp15, gp75/tyrosine-related protein-1 (“gp75/TRP-1”), humanchorionic gonadotropin (“hCG”), heparanase, HER2, human mammary tumorvirus (“HMTV”), 70 kiloDalton heat-shock protein (“HSP70”), humantelomerase reverse transcriptase (“hTERT”), insulin-like growth factorreceptor-1 (“IGFR-1”), interleukin-13 receptor (“IL-13R”), induciblenitric oxide synthase (“iNOS”), Ki67, KIAA0205, K-ras, H-ras, N-ras,KSA, LKLR-FUT, melanoma antigen-encoding gene 1 (“MAGE-1”), melanomaantigen-encoding gene 2 (“MAGE-2”), melanoma antigen-encoding gene 3(“MAGE-3”), melanoma antigen-encoding gene 4 (“MAGE-4”), mammaglobin,MAP17, Melan-A/melanoma antigen recognized by T-cells-1 (“MART-1”),mesothelin, MIC A/B, MT-MMPs, mucin, testes-specific antigen NY-ESO-1,osteonectin, p15, P170/MDR1, p53, p97/melanotransferrin, PAI-1,platelet-derived growth factor (“PDGF”), pPA, PRAME, probasin,progenipoietin, prostate-specific antigen (“PSA”), prostate-specificmembrane antigen (“PSMA”), RAGE-1, Rb, RCAS1, SART-1, SSX-family, STAT3,STn, TAG-72, transforming growth factor-alpha (“TGF-α”), transforminggrowth factor-beta (“TGF-β”), Thymosin-beta-15, tumor necrosisfactor-alpha (“TNF-α”), TP1, TRP-2, tyrosinase, vascular endothelialgrowth factor (“VEGF”), ZAG, p16INK4, and glutathione-S-transferase(“GST”).

Embodiment 13 is the method of any one of Embodiments 1-12, wherein theantibody is an antibody approved for the treatment of a cancer patient.

Embodiment 14 is the method of any one of Embodiments 1-13, wherein theantibody is selected from the group consisting of: Anti-CD20 (e.g.,rituximab; ofatumumab; tositumomab), Anti-CD52 (e.g., alemtuzumabCampath®), Anti-EGFR (e.g., cetuximab Erbitux®, panitumumab), Anti-CD2(e.g., Siplizumab), Anti-CD37 (e.g., BI836826), Anti-CD123 (e.g.,JNJ-56022473), Anti-CD30 (e.g., XmAb2513), Anti-CD38 (e.g., daratumumabDarzalex®), Anti-PDL1 (e.g., avelumab, atezolilzumab, durvalumab),CTLA-4 (e.g., ipilumumab), Anti-GD2 (e.g., 3F8, ch14.18, KW-2871,dinutuximab), Anti-CEA, Anti-MUC1, Anti-FLT3, Anti-CD19, Anti-CD40,Anti-SLAMF7, Anti-CCR4, Anti-B7-H3, Anti-ICAM1, Anti-CSF1R, anti-CA125(e.g. Oregovomab), anti-FRα (e.g. MOv18-IgG1, Mirvetuximab soravtansine(IMGN853), MORAb-202), anti-mesothelin (e.g. MORAb-009), and Anti-HER2

Embodiment 15 is the method of any one of Embodiments 1-14, wherein theantibody is specific to the HER2 antigen.

Embodiment 16 is the method of any one of Embodiments 1-15, wherein theantibody is selected from Pertuzumab, Trastuzumab, Herzuma, ABP 980, andAdo-trastuzumab emtansine.

Embodiment 17 is the method of any one of Embodiments 1-16, wherein thefirst and/or second TAA has been modified to prevent binding of theantibody to the first and/or second TAA.

Embodiment 18 is the method of any one of Embodiments 1-17, wherein thefirst TAA is HER2.

Embodiment 19 is the method of Embodiment 18, wherein one or more aminoacids of the HER2 are mutated to prevent the HER2 antibody from bindingthe HER2 antigen.

Embodiment 20 is the method of any one of Embodiments 18-19, wherein theone or more amino acids of HER2 are mutated to prevent extracellulardimerization, tyrosine kinase activity, and/or phosphorylation of theHER2 polypeptide and/or antigen.

Embodiment 21 is the method of any one of Embodiments 18-20, wherein oneor more mutations have been made to at least one of 3 loops in ajuxtamembrane region of HER2.

Embodiment 22 is the method of any one of 18-21, wherein the 3 loops ofthe juxtamembrane region of HER2 is selected from amino acids 579-583(loop1), 592-595 (loop2), and 615-625 (loop3).

Embodiment 23 is the method of any one of Embodiments 18-22, wherein theHER2 antigen comprises at least one mutation in at least one of aminoacids H267, Y274, S310, L317, H318, K333, E580, D582, P594, F595, K615,and Q624.

Embodiment 24 is the method of any one of Embodiments 18-23, wherein theHER2 antigen comprises at least one mutation selected from the groupconsisting of: E580A, F595A, K615A, S310A, 1318A, L317A, D277R, E280K,K573M, and Y1023A.

Embodiment 25 is the method of any one of Embodiments 18-24, wherein theHER2 antigen comprises at least one mutation to amino acids 1139-1248.

Embodiment 26 is the method of any one of Embodiments 18-25, wherein theHER2 antigen comprises at least one deletion to amino acids 1139-1248.

Embodiment 27 is the method of any one of Embodiments 18-26, wherein theHER2 antigen comprises a deletion of amino acids 1139-1248.

Embodiment 28 is the method of any one of Embodiments 18-27, wherein thefirst nucleic acid encodes a HER2 antigen having at least 90%, 95%, 97%98%, or 99% identity to SEQ ID NO:1.

Embodiment 29 is the method of any one of Embodiments 18-28, wherein thefirst nucleic acid encodes a HER2 antigen comprising SEQ ID NO:1.

Embodiment 30 is the method of any one of Embodiments 18-29, wherein thefirst nucleic acid encodes a HER2 antigen, the first nucleic acid havingat least 90%, 95%, 97% 98%, or 99% identity to SEQ ID NO:2.

Embodiment 31 is the method of any one of Embodiments 18-30, wherein thefirst nucleic acid comprises SEQ ID NO:2.

Embodiment 32 is the method of any one of Embodiments 1-31, wherein theMVA comprises a second nucleic acid encoding a second heterologous TAAdifferent from the first TAA.

Embodiment 33 is the method of Embodiment 32, wherein the second TAA isBrachyury.

Embodiment 34 is the method of Embodiment 33, wherein the Brachyuryantigen comprises one or more mutations that prevent nuclearlocalization of the Brachyury antigen.

Embodiment 35 is the method of any one of Embodiments 33-34, wherein theBrachyury antigen comprises one or more mutations to the nuclearlocalization signal (NLS) domain.

Embodiment 36 is the method of any one of Embodiments 33-35, wherein theBrachyury antigen has the NLS domain deleted.

Embodiment 37 is the method of any one of Embodiments 33-36, wherein thesecond nucleic acid comprises a Brachyury antigen having at least 90%,95%, 97% 98%, or 99% identity to SEQ ID NO:3.

Embodiment 38 is the method of any one of Embodiments 33-37, wherein thesecond nucleic acid comprises SEQ ID NO:3.

Embodiment 39 is the method of any one of Embodiments 33-38, wherein thesecond nucleic acid encodes a Brachyury antigen, the second nucleic acidhaving at least 90%, 95%, 97% 98%, or 99% identity to SEQ ID NO:4.

Embodiment 40 is the method of any one of Embodiments 33-39, wherein 10the second nucleic acid comprises SEQ ID NO:4.

Embodiment 41 is the method of any one of Embodiments 1-40, wherein theantibody is selected from the group consisting of: Anti-CD20 (rituximab;ofatumumab; tositumomab), Anti-CD52 (alemtuzumab Campath®), Anti-EGFR(cetuximab Erbitux®, panitumumab), Anti-CD38 (daratumumab Darzalex®),Anti-PDL1 (avelumab), Anti-CTLA4 (ipilimumab), Anti-GD2 (3F8, ch14.18,KW-2871, dinutuximab), Anti-CEA, Anti-Muel, Anti-FLT3L, Anti-CD19,Anti-CD40, Anti-SLAMF7, Anti-CCR4, Anti-B7-H3, Anti-ICAM1, Anti-CSF1R,and Anti-HER2.

Embodiment 42 is the method of any one of Embodiments 1-41, wherein theantibody is specific to the HER2 antigen.

Embodiment 43 is the method of Embodiment 42, wherein the anti-HER2antibody is selected from Pertuzumab, Trastuzumab, and Ado-trastuzumabemtansine.

Embodiment 44 the method of any one of Embodiments 1-43, wherein theantibody and the MVA are administered as a homologous prime-boostregimen.

Embodiment 45 is the method of any one of Embodiments 1-44, wherein theMVA is MVA-BN, or a derivative of MVA-BN.

Embodiment 46 is the method of any one of Embodiments 1-45, wherein thecancer patient is suffering from and/or has been diagnosed with a cancerselected from the group consisting of breast cancer, lung cancer, headand neck cancer, thyroid, melanoma, gastric cancer, bladder cancer,kidney cancer, liver cancer, melanoma, pancreatic cancer, prostatecancer, ovarian cancer, or colorectal cancer.

Embodiment 47 is a pharmaceutical combination for use in reducing tumorsize and/or increasing survival in a cancer patient, the combinationcomprising: a) a recombinant modified vaccinia virus Ankara (MVA)comprising a first nucleic acid encoding a first heterologoustumor-associated antigen (TAA) that when administered intravenouslyinduces both an enhanced Natural Killer (NK) cell response and anenhanced T cell response as compared to an NK cell response and a T cellresponse induced by a non-intravenous administration of a recombinantMVA virus comprising a nucleic acid encoding a heterologous TAA; and b)an antibody, wherein the antibody comprises an Fc domain and is specificto an antigen that is expressed on the cell membrane of a tumor cell;wherein administration of a) and b) to the cancer patient reduces tumorsize and/or increases the survival rate of the cancer patient ascompared to an non-IV administration of a) or an administration of b)alone.

Embodiment 48 is a pharmaceutical combination for use in reducing tumorsize and/or increasing survival in a cancer patient, the combinationcomprising: a) a recombinant modified vaccinia virus Ankara (MVA)comprising a first nucleic acid encoding a first heterologoustumor-associated antigen (TAA) that when administered intravenouslyinduces both an enhanced Natural Killer (NK) cell response and anenhanced T cell response as compared to an NK cell response and a T cellresponse induced by a non-intravenous administration of a recombinantMVA comprising a nucleic acid encoding a heterologous TAA; and b) anantibody, wherein the antibody comprises an Fc domain and is specific toan antigen that is expressed on the cell membrane of a tumor cell thatwhen the antibody is administered, the antibody binds to the antigen onthe tumor cell in the human cancer patient and induces antibodydependent cell-mediated cytotoxicity (ADCC); wherein administration ofa) and b) to the cancer patient reduces tumor size and/or increases thesurvival rate of the cancer patient as compared to an non-IVadministration of a) or an administration of b) alone.

Embodiment 49 is the pharmaceutical combination of any one ofEmbodiments 47-48, wherein the CD40L is encoded by the recombinant MVA.

Embodiment 50 is the pharmaceutical combination of any one ofEmbodiments 47-49, wherein the recombinant MVA further comprises asecond nucleic acid encoding a second heterologous TAA.

Embodiment 51 is the pharmaceutical combination of any one ofEmbodiments 47-50, wherein the first and/or second TAA is selected fromthe group consisting of: 5-α-reductase, α-fetoprotein (“AFP”), AM-1,APC, April, B melanoma antigen gene (“BAGE”), β-catenin, Bc112, bcr-abl,Brachyury, CA-125, caspase-8 (“CASP-8”, also known as “FLICE”),Cathepsins, CD19, CD20, CD21/complement receptor 2 (“CR2”), CD22/BL-CAM,CD23/FesRII, CD33, CD35/complement receptor 1 (“CR1”), CD44/PGP-1,CD45/leucocyte common antigen (“LCA”), CD46/membrane cofactor protein(“MCP”), CD52/CAMPATH-1, CD55/decay accelerating factor (“DAF”),CD59/protectin, CDCl₂7, CDK4, carcinoembryonic antigen (“CEA”), c-myc,cyclooxygenase-2 (“cox-2”), deleted in colorectal cancer gene (“DCC”),DcR3, E6/E7, CGFR, EMBP, Dna78, farnesyl transferase, fibroblast growthfactor-8a (“FGF8a”), fibroblast growth factor-8b (“FGF8b”), FLK-1/KDR,folic acid receptor, G250, G melanoma antigen gene family(“GAGE-family”), gastrin 17, gastrin-releasing hormone, ganglioside 2(“GD2”)/ganglioside 3 (“GD3”)/ganglioside-monosialic acid-2 (“GM2”),gonadotropin releasing hormone (“GnRH”), UDP-GlcNAc:R₁Man(α1-6)R2[GlcNAc to Man(α1-6)] β1,6-N-acetylglucosaminyltransferase V (“GnT V”),GP1, gp100/Pme117, gp-100-in4, gp15, gp75/tyrosine-related protein-1(“gp75/TRP-1”), human chorionic gonadotropin (“hCG”), heparanase, HER2,human mammary tumor virus (“HMTV”), 70 kiloDalton heat-shock protein(“HSP70”), human telomerase reverse transcriptase (“hTERT”),insulin-like growth factor receptor-1 (“IGFR-1”), interleukin-13receptor (“IL-13R”), inducible nitric oxide synthase (“iNOS”), Ki67,KIAA0205, K-ras, H-ras, N-ras, KSA, LKLR-FUT, melanoma antigen-encodinggene 1 (“MAGE-1”), melanoma antigen-encoding gene 2 (“MAGE-2”), melanomaantigen-encoding gene 3 (“MAGE-3”), melanoma antigen-encoding gene 4(“MAGE-4”), mammaglobin, MAP17, Melan-A/melanoma antigen recognized byT-cells-1 (“MART-1”), mesothelin, MIC A/B, MT-MMPs, mucin,testes-specific antigen NY-ESO-1, osteonectin, p15, P170/MDR1, p53,p97/melanotransferrin, PAI-1, platelet-derived growth factor (“PDGF”),pPA, PRAME, probasin, progenipoietin, prostate-specific antigen (“PSA”),prostate-specific membrane antigen (“PSMA”), RAGE-1, Rb, RCAS1, SART-1,SSX-family, STAT3, STn, TAG-72, transforming growth factor-alpha(“TGF-α”), transforming growth factor-beta (“TGF-β”), Thymosin-beta-15,tumor necrosis factor-alpha (“TNF-α”), TP1, TRP-2, tyrosinase, vascularendothelial growth factor (“VEGF”), ZAG, p16INK4, andglutathione-S-transferase (“GST”)

Embodiment 52 is the pharmaceutical combination of any one ofEmbodiments 47-51, wherein the first TAA is HER2.

Embodiment 53 is the pharmaceutical combination of Embodiment 52,wherein the HER2 antigen comprises at least one mutation selected fromthe group consisting of: E580A, F595A, K615A, S310A, H318A, L317A,D277R, E280K, K573M, and Y1023A.

Embodiment 54 is the pharmaceutical combination of any one ofEmbodiments 52-53, wherein the first nucleic acid comprises a HER2antigen having at least 90%, 95%, 97% 98%, or 99% identity to SEQ IDNO:1.

Embodiment 55 is the pharmaceutical combination of any one ofEmbodiments 52-54, wherein the first nucleic acid comprises SEQ ID NO:1.

Embodiment 56 is the pharmaceutical combination of any one ofEmbodiments 52-55, wherein the first nucleic acid encodes a HER2antigen, the first nucleic acid having at least 90%, 95%, 97% 98%, or99% identity to SEQ ID NO:2.

Embodiment 57 is the pharmaceutical combination of any one ofEmbodiments 52-56, wherein the first nucleic acid comprises SEQ ID NO:2.

Embodiment 58 is the pharmaceutical combination of any one ofEmbodiments 47-57, wherein the MVA comprises a second nucleic acidencoding a second heterologous TAA different from the first TAA.

Embodiment 59 is the pharmaceutical combination of Embodiment 58,wherein the second TAA is Brachyury.

Embodiment 60 is the pharmaceutical combination of Embodiment 59,wherein the second nucleic acid comprises a Brachyury antigen having atleast 90%, 95%, 97% 98%, or 99% identity to SEQ ID NO:3.

Embodiment 61 is the pharmaceutical combination of any one ofEmbodiments 59-60, wherein the second nucleic acid encodes an antigencomprising SEQ ID NO:3.

Embodiment 62 is the pharmaceutical combination of any one ofEmbodiments 59-61, wherein the second nucleic acid encodes a Brachyuryantigen, the second nucleic acid having at least 90%, 95%, 97% 98%, or99% identity to SEQ ID NO:4.

Embodiment 63 is the pharmaceutical combination of any one of 5Embodiments 59-62, wherein the second nucleic acid comprises SEQ IDNO:4.

Embodiment 64 is the pharmaceutical combination of any one ofEmbodiments 47-63, wherein the antibody is selected from the groupconsisting of: anti-CD20 (e.g., rituximab, ofatumumab, tositumomab),anti-CD52 (e.g., alemtuzumab, Campath® antibody), anti-EGFR (e.g.,cetuximab, Erbitux® antibody, panitumumab), anti-CD2 (e.g., siplizumab),anti-CD37 (e.g., BI836826), anti-CD123 (e.g., JNJ-56022473), anti-CD30(e.g., XmAb2513), anti-CD38 (e.g., daratumumab, Darzalex® antibody,anti-PDL1 (e.g., avelumab, atezolilzumab, durvalumab), CTLA-4 (e.g.,ipilumumab), anti-GD2 (e.g., 3F8, ch14.18, KW-2871, dinutuximab),anti-CEA, anti-MUC1, anti-FLT3, anti-CD19, anti-CD40, anti-SLAMF7,anti-CCR4, anti-B7-H3, anti-ICAM1, anti-CSF1R, anti-CA125 (e.g.,oregovomab), anti-FRα (e.g. MOv18-IgG1, mirvetuximab soravtansine(IMGN853), MORAb-202), anti-mesothelin (e.g., MORAb-009), and anti-HER2.

Embodiment 65 is the pharmaceutical combination of any one ofEmbodiments 47-64, wherein the antibody is specific to the HER2 antigen.

Embodiment 66 is the pharmaceutical combination of Embodiment 65,wherein the antibody is selected from pertuzumab, trastuzumab, Herzuma®antibody, ABP 980, and ado-trastuzumab emtansine.

Embodiment 67 is the pharmaceutical combination of any one ofEmbodiments 47-66, wherein the MVA is MVA-BN or a derivative of MVA-BN.

Embodiment 68 is a method for inducing an enhanced Natural Killer (NK)response in a cancer patient comprising administering a pharmaceuticalcombination of any one of Embodiments 47-66, wherein administering thecombination enhances the NK cell response of the cancer patient ascompared to a non-intravenously administered combination of a) or anadministration of b) alone.

Embodiment 69 is a method for inducing both an enhanced innate and anenhanced adaptive immune response in a cancer patient comprisingadministering a pharmaceutical combination of any one of Embodiments47-67 wherein administering the combination enhances the NK cellresponse of the cancer patient as compared to a non-intravenouslyadministered combination of (a) or an administration of (b) alone.

Embodiment 70 is the method of Embodiment 69, wherein the enhancedadaptive immune response comprises an enhanced T cell response.

Embodiment 71 is the method of Embodiment 70, wherein the enhanced Tcell response comprises an enhanced CD8 T cell response and an enhancedCD4 T cell response.

Embodiment 72 is a method for enhancing antibody therapy in a cancerpatient, the method comprising administering a pharmaceuticalcombination of any one of Embodiments 47-67, wherein administering thepharmaceutical combination enhances antibody dependent cell-mediatedcytotoxicity (ADCC) induced by the antibody therapy, as compared toadministering the antibody therapy alone.

Embodiment 73 is a method for enhancing the killing of tumor cellshaving reduced MHC levels in a cancer patient, the method comprisingadministering a pharmaceutical combination of any one of Embodiments47-67, wherein administering the combination enhances the killing oftumor cells having reduced MHC levels as compared to administering b)alone.

Embodiment 74 is a method for treating a human cancer patient comprisingadministering to the cancer patient a pharmaceutical combination of anyone of Embodiments 47-67.

Embodiment 75 is a method of any one of Embodiments 68-74, wherein therecombinant MVA is administered at the same time or after the antibody.

Embodiment 76 is a method of any one of Embodiments 68-74, wherein therecombinant MVA is administered after the antibody.

Embodiment 77 is a method of any one of Embodiments 68-75, wherein therecombinant MVA is administered 0 to 8 days, 0 to 7 days, 0 to 6 days, 0to 5 days, 0 to 4 days, 0 to 3 days, 0 to 2 days, or 0 to 1 day afterthe antibody.

Embodiment 78 is a method of any one of Embodiments 68-75, wherein therecombinant MVA is administered 1 to 8 days, 1 to 7 days, 1 to 6 days, 1to 5 days, 1 to 4 days, 1 to 3 days, 1 to 2 days, or 1 day after theantibody.

Embodiment 79 is a method of any one of Embodiments 68-77, wherein therecombinant MVA is administered 0 to 3 days, 0 to 2 days, 0 to 1 dayafter the antibody.

Embodiment 80 is a method of Embodiment 79, wherein the recombinant MVAis administered 0 to 3 days after the antibody.

Embodiment 81 is a method of any one of Embodiments 68-80, wherein theantibody and the MVA are administered as part of a homologous orheterologous prime boost regimen.

Embodiment 82 is a method of any one of Embodiments 68-80, wherein theantibody and the MVA are administered as part of a homologous primeboost regimen.

Embodiment 83 is a method of any one of Embodiments 1-45 and 68-82,wherein the cancer patient is suffering from and/or is diagnosed with acancer selected from the group consisting of: breast cancer, lungcancer, head and neck cancer, thyroid, melanoma, gastric cancer, bladdercancer, kidney cancer, liver cancer, melanoma, pancreatic cancer,prostate cancer, ovarian cancer, or colorectal cancer.

Embodiment 84 is a pharmaceutical combination for use in reducing tumorvolume and/or increasing survival of a cancer patient, thepharmaceutical combination comprising the pharmaceutical combination ofany one of Embodiments 47-67, wherein administering the combinationreduces tumor volume and/or increases survival of the patient, ascompared to a non-intravenously administered combination of (a) or anadministration of (b) alone.

Embodiment 85 is use of the pharmaceutical combination of any one ofEmbodiments 47-67 in a method of reducing tumor volume and/or increasingsurvival of a cancer patient.

Embodiment 86 is use of the pharmaceutical combination of any one ofEmbodiments 47-67 in the preparation of a pharmaceutical or medicamentfor reducing tumor volume and/or increasing survival of a cancerpatient.

Embodiment 87 is a method of reducing tumor size and/or increasingsurvival in a cancer patient, the method comprising: (a) intravenouslyadministering to the cancer patient a recombinant modified vacciniavirus Ankara (MVA) comprising a first nucleic acid encoding a firstheterologous tumor-associated antigen (TAA) that when administeredintravenously induces both an enhanced innate immune response and anenhanced T cell response as compared to an innate immune response and aT cell response induced by a non-intravenous administration of arecombinant MVA comprising a first nucleic acid encoding a firstheterologous cancer-associated antigen; and (b) administering to thecancer patient an antibody, wherein the antibody comprises an Fc domainand is specific to an antigen that is expressed on the cell membrane ofa tumor cell; wherein administration of (a) and (b) to the cancerpatient reduces tumor size in the cancer patient and/or increases thesurvival rate of the cancer patient as compared to a non-intravenousadministration of a) or an administration of b) alone.

Embodiment 88 is the method of Embodiment 87, wherein the enhancedinnate immune response comprises at least one of an enhanced NK cellresponse, an enhanced macrophage response, an enhanced monocyteresponse, an enhanced neutrophil response, an enhanced basophilresponse, an enhanced eosinophil response, an enhanced mast cellresponse, and an enhanced dendritic cell response.

Embodiment 89 is the method of any one of Embodiments 87-88, wherein therecombinant MVA further encodes CD40L.

Embodiment 90 is the method of any one of Embodiments 88-89, whereininducing an enhanced NK cell response comprises at least one of (a)inducing an enhanced ADCC response and (b) inducing NK cells to targetand kill tumor cells having low MHC expression.

Embodiment 91 is the method of any one of Embodiments 87-90, wherein therecombinant MVA is administered at the same time or after the antibody.

Embodiment 92 is the method of any one of Embodiments 87-91, wherein therecombinant MVA is administered after the antibody.

Embodiment 93 is the method of any one of Embodiments 87-92, wherein theMVA comprises a first and/or second nucleic acid encoding a first and/orsecond heterologous TAA from any one of Embodiments 51-63.

Embodiment 94 is the method of any one of Embodiments 87-93, wherein theantibody comprises an antibody from any one of Embodiments 64-66.

Embodiment 95 is a pharmaceutical combination for reducing tumor sizeand/or increasing survival in a cancer patient, the combinationcomprising: (a) a recombinant modified vaccinia virus Ankara (MVA)comprising a first nucleic acid encoding a first heterologoustumor-associated antigen (TAA) that when administered intravenouslyinduces both an enhanced innate immune response and an enhanced T cellresponse as compared to an innate immune response and a T cell responseinduced by a non-intravenous administration of a recombinant MVA viruscomprising a nucleic acid encoding a heterologous TAA; and (b) anantibody, wherein the antibody comprises an Fc domain and is specific toan antigen that is expressed on the cell membrane of a tumor cell;wherein administration of (a) and (b) to the cancer patient reducestumor size and/or increases the survival rate of the cancer patient ascompared to an non-IV administration of a) or an administration of b)alone.

Embodiment 96 is the pharmaceutical combination of Embodiment 95,wherein the recombinant MVA further encodes CD40L.

Embodiment 97 is the pharmaceutical combination of any one ofEmbodiments 95-96, wherein the enhanced innate immune response comprisesat least one of an enhanced NK cell response, an enhanced macrophageresponse, an enhanced neutrophil response, an esophil response, anenhanced eosinophil response, an enhanced mast cell response, and anenhanced dendritic cell response.

Embodiment 98 is the pharmaceutical combination of any one ofEmbodiments 95-97, wherein inducing an enhanced NK cell responsecomprises at least one of (a) inducing an enhanced ADCC response and (b)inducing NK cells to target and kill tumor cells having low MHCexpression.

Embodiment 99 is the pharmaceutical combination of any one ofEmbodiments 95-98, wherein the MVA comprises a first and/or secondnucleic acid encoding a first and/or second heterologous TAA from anyone of Embodiments 51-63.

Embodiment 100 is the pharmaceutical combination of any one ofEmbodiments 95-99, wherein the antibody comprises an antibody from anyone of Embodiments 64-66.

Embodiment 101 is a pharmaceutical combination for reducing tumor sizeand/or increasing survival in a cancer patient, the combinationcomprising: (a) a recombinant poxvirus comprising a first nucleic acidencoding a first heterologous tumor-associated antigen (TAA) that whenadministered intravenously induces both an enhanced Natural Killer (NK)cell response and an enhanced T cell response as compared to an NK cellresponse and a T cell response induced by a non-intravenousadministration of a recombinant poxvirus comprising a nucleic acidencoding a heterologous TAA; and (b) an antibody, wherein the antibodycomprises an Fe domain and is specific to an antigen that is expressedon the cell membrane of a tumor cell; wherein administration of (a) and(b) to the cancer patient reduces tumor size and/or increases thesurvival rate of the cancer patient as compared to an non-IVadministration of (a) or an administration of (b) alone.

Embodiment 102 is the pharmaceutical combination of Embodiment 101,wherein the recombinant poxvirus further encodes CD40L.

Embodiment 103 is the pharmaceutical combination of any one ofEmbodiments 101-102, wherein the MVA comprises a first and/or secondnucleic acid encoding a first and/or second heterologous TAA from anyone of Embodiments 51-63.

Embodiment 104 is the pharmaceutical combination of any one ofEmbodiments 101-103, wherein the antibody comprises an antibody from anyone of Embodiments 64-66.

Embodiment 105 is a method of reducing tumor size and/or increasingsurvival in a cancer patient, the method comprising: (a) intravenouslyadministering to the cancer patient a recombinant modified VacciniaAnkara (MVA) comprising a first nucleic acid encoding a firstheterologous tumor-associated antigen (TAA) that when administeredintravenously induces both an enhanced Natural Killer (NK) cell responseand an enhanced T cell response as compared to an NK cell response and aT cell response induced by a non-intravenous administration of arecombinant MVA virus comprising a nucleic acid encoding a heterologoustumor-associated antigen; and (b) administering to the cancer patient anantibody, wherein the antibody is specific to an antigen that isexpressed on the cell membrane of a tumor cell; wherein administrationof (a) and (b) to the cancer patient reduces tumor size in the cancerpatient and/or increases the survival rate of the cancer patient ascompared to an non-intravenous administration of (a) or anadministration of (b) alone.

Embodiment 106 is a pharmaceutical combination for reducing tumor sizeand/or increasing survival in a cancer patient, the combinationcomprising: (a) an antibody, wherein the antibody comprises an Fc domainand is specific to an antigen that is expressed on the cell membrane ofa tumor cell that when administered to the patient induces ADCC in thepatient; and (b) a recombinant modified Vaccinia Ankara (MVA) viruscomprising a first nucleic acid encoding a first heterologoustumor-associated antigen (TAA), wherein the MVA when administeredintravenously to the patient induces an enhanced Natural Killer (NK)cell response that enhances the ADCC in the patient and enhances NK cellmediated toxicity, and wherein the MVA induces an enhanced T cellresponse; as compared to an NK cell response and a T cell responseinduced by a non-intravenous administration of a recombinant MVA viruscomprising a nucleic acid encoding a heterologous tumor-associatedantigen; and wherein administration of (a) and (b) to the cancer patientreduces tumor size and/or increases the survival rate of the cancerpatient as compared to an non-IV administration of (a) or anadministration of (b) alone.

Embodiment 107 is the pharmaceutical combination of Embodiment 106,wherein the recombinant MVA further comprises a nucleic acid encodingCD40L.

Embodiment 108 is the pharmaceutical combination of any one ofEmbodiments 106-107, wherein the MVA comprises a first and/or secondnucleic acid encoding a first and/or second heterologous TAA from anyone of Embodiments 51-63.

Embodiment 109 is a method for reducing tumor volume and/or increasingsurvival in a cancer patient comprising administering the pharmaceuticalcombination of any one of Embodiments 106-108.

Embodiment 110 is a pharmaceutical combination for use in reducing tumorvolume and/or increasing survival of a cancer patient, thepharmaceutical combination comprising the pharmaceutical combination ofany one of Embodiments 106-108, wherein administering the combinationreduces tumor volume and/or increase survival of the patient, ascompared to compared to an administration of a) or a non-intravenousadministration of b) alone.

Embodiment 111 is use of the pharmaceutical combination of any one ofEmbodiments 106-108 in a method of reducing tumor volume and/orincreasing survival of a cancer patient.

Embodiment 112 is use of the pharmaceutical combination of any one ofEmbodiments 106-108 for the preparation of a pharmaceutical ormedicament for reducing tumor volume and/or increasing survival of acancer patient.

Embodiment 113 is a method for increasing the effectiveness of antibodytherapy in a cancer patient, the method comprising administering thepharmaceutical combination of any one of Embodiments 47-67, and 106-108,wherein administering the combination to the patient decreases theantibody concentration needed for NK cell-mediated toxicity in tumorcells.

Embodiment 114 is the pharmaceutical combination of any one ofEmbodiments 47-67 and 106-108, wherein the recombinant MVA comprises anucleic acid encoding CD40L, the CD40L comprising SEQ ID NO:11.

Embodiment 115 is the pharmaceutical combination of Embodiment 114,wherein the nucleic acid encoding CD40L is a nucleic acid having atleast 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO:12.

Embodiment 116 is the method of any one of Embodiments 1-46, wherein therecombinant MVA comprises a nucleic acid encoding CD40L, the CD40Lcomprising SEQ ID NO:11.

Embodiment 117 is the method of Embodiment 116, wherein the nucleic acidencoding CD40L is a nucleic acid having at least 95%, 98%, 99%, or 100%sequence identity to SEQ ID NO:12.

Embodiment 118 is the method of Embodiment 83, wherein the Breast Canceris a HER2 overexpressing breast cancer.

Embodiment 119 is a method of any one of embodiments 18-19, wherein theone or more amino acids of HER2 are mutated to prevent extracellulardimerization, tyrosine kinase activity, and/or phosphorylation of theHER2 polypeptide and/or antigen once expressed by the recombinant MVA.

Embodiment 120 is a method of any one of embodiments 18-19, wherein theHER antigen comprises one or more mutations for preventing the HER2antibody from binding the HER2 antigen expressed by the recombinant MVA.

Embodiment 121 is a pharmaceutical combination for use of any one ofembodiments 50-63, wherein the first and/or second TAA comprises one ormore mutations to prevent binding of the antibody to the first and/orsecond TAA.

Embodiment 122 is the method of any one of Embodiments 18-20, whereinone or more mutations have been made to at least one of 3 loops in ajuxtamembrane region of HER2.

Embodiment 123 is the method of any one of 18-21, wherein the 3 loops ofthe juxtamembrane region of HER2 is selected from amino acids 579-583(loop1), 592-595 (loop2), and 615-625 (loop3).

Embodiment 124 is the method of any one of Embodiments 18-22, whereinthe HER2 antigen comprises at least one mutation in at least one ofamino acids E580A, F595A, K615A, H267A, F279A, V308A, S310A, L317A,H318A, K333A, P337A.

Embodiment 125 is the method of any one of Embodiments 18-23, whereinthe HER2 antigen comprises at least one mutation selected from the groupconsisting of: E580A, F595A, K615A, 1267A, F279A, V308A, S310A, L317A,1318A, K333A, P337A, D277R, E280K, K753M, and Y1023A.

Embodiment 126 is a pharmaceutical combination for use in therapy byreducing tumor volume and/or increasing survival of a cancer patient(preferably wherein the cancer patient is suffering from and/or isdiagnosed with a cancer selected from the group consisting of: breastcancer, lung cancer, head and neck cancer, thyroid, melanoma, gastriccancer, bladder cancer, kidney cancer, liver cancer, melanoma,pancreatic cancer, prostate cancer, ovarian cancer, or colorectalcancer), the pharmaceutical combination comprising the pharmaceuticalcombination of any one of Embodiments 47-67, wherein administering thecombination reduces tumor volume and/or increases survival of thepatient, as compared to an non-intravenously administered combination of(a) or an administration of (b) alone.

Embodiment 127 is use of the pharmaceutical combination of any one ofEmbodiments 47-67 in the preparation of a pharmaceutical or medicamentfor reducing tumor volume and/or increasing survival of a cancerpatient, preferably wherein the cancer patient is suffering from and/oris diagnosed with a cancer selected from the group consisting of: breastcancer, lung cancer, head and neck cancer, thyroid, melanoma, gastriccancer, bladder cancer, kidney cancer, liver cancer, melanoma,pancreatic cancer, prostate cancer, ovarian cancer, or colorectalcancer.

Embodiment 128 is the method of any one of Embodiments 18-27, whereinthe first nucleic acid encodes a HER2 antigen having at least 90%, 95%,97% 98%, or 99% identity to SEQ ID NO: 13.

Embodiment 129 is the method of any one of Embodiments 18-28, whereinthe first nucleic acid encodes a HER2 antigen comprising SEQ ID NO: 13.

Embodiment 130 is the method of any one of Embodiments 18-29, whereinthe first nucleic acid encodes a HER2 antigen, the first nucleic acidhaving at least 90%, 95%, 97% 98%, or 99% identity to SEQ ID NO: 14.

Embodiment 131 is the method of any one of Embodiments 18-30, whereinthe first nucleic acid comprises SEQ ID NO: 14.

Embodiment 132 is the method of any one of Embodiments 18-27, whereinthe first nucleic acid encodes a HER2 antigen having at least 90%, 95%,97% 98%, or 99% identity to the full length of SEQ ID NO: 13.

Embodiment 133 is the method of any one of Embodiments 18-29, whereinthe first nucleic acid encodes a HER2 antigen, the first nucleic acidhaving at least 90%, 95%, 97% 98%, or 99% identity to the full length ofSEQ ID NO: 14.

Embodiment 134 is the method of any one of Embodiments 18-27, whereinthe first nucleic acid encodes a HER2 antigen having at least 90%, 95%,97% 98%, or 99% identity to the full length of SEQ ID NO: 1.

Embodiment 135 is the method of any one of Embodiments 18-29, whereinthe first nucleic acid encodes a HER2 antigen, the first nucleic acidhaving at least 90%, 95%, 97% 98%, or 99% identity to the full length ofSEQ ID NO: 2.

Embodiment 136 is the method of any one of Embodiments 34-36, whereinthe first nucleic acid encodes a HER2 antigen having at least 90%, 95%,97% 98%, or 99% identity to the full length of SEQ ID NO: 3.

Embodiment 137 is the method of any one of Embodiments 18-29, whereinthe first nucleic acid encodes a HER2 antigen, the first nucleic acidhaving at least 90%, 95%, 97% 98%, or 99% identity to the full length ofSEQ ID NO: 4.

EXAMPLES

The following examples illustrate the invention but should not beconstrued as in any way limiting the scope of the claims.

Example 1: Intravenous Administration of Recombinant MVA Results inStronger Activation of NK Cells

C57BL/6 mice were immunized subcutaneously (SC) or intravenously (IV)with 5×10⁷ TCID₅₀ MVA-OVA (shown as rMVA) or MVA-OVA-CD40L (shown asrMVA-CD40L). PBS was injected SC. One day later, NK Cell frequencies andprotein expression (shown as Geometric Mean Fluorescence Intensity(GMFI)) were assessed using flow cytometry in the spleen (shown in FIGS.1A-1G), in the liver shown in (FIGS. 2A-2G), and in the lung (shown inFIGS. 3A-3G) by staining for (A) NKp46⁺ CD3⁻ cells; (B) CD69; (C) NKG2D;(D) FasL; (E); Bcl-X_(L); (F), CD70; and (G) IFN-γ.

Additionally, C57BL/6 mice were immunized subcutaneously (SC) orintravenously (IV) with 5×10⁷ TCID₅₀ of a recombinant MVA encoding HER2,TWIST, and CD40L antigens (shown as MVA-HER2-TWIST-CD40L). PBS wasinjected SC. One day later, NK Cell frequencies and protein expression(shown as Geometric Mean Fluorescence Intensity (GMFI)) were assessedusing flow cytometry in the spleen (shown FIGS. 4A-4F), in the liver(shown in FIGS. 5A-5F), and in the lung (shown in FIGS. 6A-6F) bystaining for (A) NKp46⁺ CD3⁻ cells; (B) CD69; (C) FasL; (D); Bcl-X_(L);(E), CD70; and (F) IFN-γ.

Shown in the Figures, splenic NK cell frequencies dropped or weremaintained after rMVA, rMVA-CD40L, and MVA-HER2-TWIST-CD40L injectionregardless of the application route. In (A) IV rMVA applicationincreased NK cell frequencies in liver and lung as compared to SCapplication. In (B) CD69 is a stimulatory receptor for NK cells (Borregoet al. (1999) Immunology 97: 159-65) and is strongly upregulated afterIV but not SC injection of rMVA, rMVA-CD40L, and MVA-HER2-TWIST-CD40L.The highest CD69 expression was induced by rMVA-CD40L IV application. InFIGS. 1-3(C) the activating C-type lectin-like receptor NKG2D isupregulated on NK cells after rMVA and rMVA-CD40L immunization ascompared to PBS treatment. In FIGS. 1-3(D) and FIGS. 4-6(C) theapoptosis-inducing factor FasL (CD95L) is upregulated on NK cells afterrMVA and rMVA-CD40L immunization as compared to PBS treatment. In FIGS.1-3(D) and FIGS. 4-6(C) spleen and lung, FasL expression was highestafter IV rMVA-CD40L and MVA-HER2-TWIST-CD40L injection. In FIGS. 1-3(E)and 4-6(D) IV rMVA-CD40L and MVA-HER2-TWIST-CD40L immunization also leadto a higher expression of the anti-apoptotic Bel family member Bcl-x_(L)as compared to SC immunization. In FIGS. 1-3(F) and 4-6 (E) upregulationof the co-stimulatory molecule CD70, a member of the tumor necrosisfactor (TNF) superfamily, is induced by IV injection of rMVA,rMVA-CD40L, and MVA-HER2-TWIST-CD40L, especially on splenic NK cells. InFIGS. 1-3(G) and 4-6(F) importantly, the effector cytokine IFN-γ is moststrongly expressed after IV rMVA-CD40L or IV MVA-HER2-TWIST-CD40Limmunization in spleen, lung and liver. These data show that IVimmunization with either rMVA-CD40L or MVA-HER2-TWIST-CD40L but not SCimmunization leads to a strong, systemic NK cell activation.

Example 2: Intravenous Administration of Recombinant MVA-CD40L Resultsin Stronger Systemic Activation of NK Cells

C57BL/6 mice were immunized IV with 5×10⁷ TCID₅₀ MVA-OVA (rMVA),MVA-OVA-CD40L (rMVA-CD40L), or PBS. Six hours after injection, serumcytokine levels (A) IFN-γ, (B) IL-12p70, and (C) CD69⁺ granzyme B werequantified by a bead assay (Luminex) (A and B) and flow cytometry (C),as shown in FIGS. 7A-7F. The NK cell activating cytokine IL-12p70 wasonly detectable after rMVA-CD40L immunization. The concentration ofIFN-γ was higher after rMVA-CD40L as compared to rMVA immunization. Theincreased serum levels of IFN-γ are in line with higher GMFI IFN-γ of NKcells (compared to FIG. 1G) and higher frequencies of spleen CD69⁺Granzyme B+NK cells 48 hours after rMVA-CD40L immunization.

Similar responses were seen in NHPs (Macacafascicularis) after IVinjection of MVA-MARV-GP-huCD40L, namely higher serum concentrations ofIFN-γ (D) and IL-12p40/70 (E) as well as more proliferating (Ki67⁺) NKcells (F) as compared to MVA-MARV-GP. These data demonstrate thatCD40L-encoding MVA vaccines have comparable immunological properties inmice and NHPs.

Example 3: Intravenous Administration Results in Stronger NK CellActivation and Proliferation Over Time

C57BL/6 mice were immunized IV with 5×10⁷ TCID₅₀ MVA-OVA (rMVA),MVA-OVA-CD40L (rMVA-CD40L), or PBS. NK cell activation and proliferationwere measured for (A) CD3⁻ CD19-NKp46, (B) Ki67, and (C) CD69 in thespleen, liver, and lung on day 1 and 4 after immunization. Results areshown in FIG. 8.

Shown in FIG. 8(A) Splenic NK cell frequencies (defined as CD3⁻CD19-NKp46) temporarily dropped by day 1 after rMVA and rMVA-CD40Limmunization as seen in Example 1, but were increased by day 4 comparedto PBS. In liver and lung both vectors induced higher NK cellfrequencies already by day 1. Unexpectedly on day 4, NK cellsfrequencies in the liver were 2.4-times higher after rMVA and 4-timeshigher after rMVA-CD40L immunization compared to PBS. Similarly, NK cellfrequencies in lung and blood were drastically enhanced on day 4 afterimmunization shown in 8A. In line with the systemic NK cell increase,more than 80% of all NK cells on day 4 after rMVA and rMVA-CD40Limmunization were in proliferation, as indicated by the expression ofthe proliferation marker Ki67 in 8B. The peak of CD69 expression (shownas Geometric Mean Fluorescence Intensity (GMFI)) in all analyzed organsand blood was on day 1. By day 4 after immunization, CD69 expression wasback at basal level (FIG. 8C).

Example 4: Intravenous Administration of Recombinant MVA Results inStronger NK Cell Mediated Toxicity

C57BL/6 mice were immunized IV with 5×10⁷ TCID₅₀ MVA-OVA (rMVA),MVA-OVA-CD40L (rMVA-CD40L), or PBS. 24 hours later mice were sacrificedand splenic NK cells were purified by magnetic cells sorting and used aseffectors in an effector: target killing assay. Briefly NK cells werecultured with CFSE-labelled MHC class I-deficient YAC-1 cells at theratios shown in FIG. 9. Specific killing was assessed by quantifyingunviable CFSE+ YAC-1 cells by flow cytometry. As a result, rMVA andrMVA-CD40L activated NK cells upon IV immunization are potent killers ofMHC class I-deficient target cells.

Example 5: Intravenous Administration of Recombinant MVA Results inEnhanced ADCC

Shown in FIG. 10A, C57BL/6 mice were treated IV either with 25 μganti-CD4 (clone GK1.5), MVA-OVA (rMVA)+5 μg rat IgG2b, or 1 μg anti-CD4or MVA-OVA (rMVA)+1 μg anti-CD4. 24 hours later mice were sacrificed andCD4 T cell (CD3⁺CD4⁺) depletion in the liver was analyzed by flowcytometry. 25 μg anti-CD4 was defined as 100% specific killing. Incomparison to rMVA+IgG2b (defined as 0% specific killing), 1 μg anti-CD4depleted 61% of all liver CD4 T cells. The combination of rMVA +1 μganti-CD4 resulted in 93% CD4 T cell depletion, which is almost asefficient as 25 μg 10 anti-CD4. Thus, combination of rMVA and depletingantibodies leads to synergistic target cell killing in vivo.

Shown in FIGS. 10B and 10C to assess ex vivo ADCC activity of NK cells,C57BL/6 (B) or Balb/c (C) mice were immunized IV either with PBS,MVA-OVA (rMVA) or MVA-OVA-CD40L (rMVA-CD40L). 24 hours later mice weresacrificed; splenic NK cells were purified by magnetic cell sorting andused as effectors in antibody-dependent effector: target killing assays.(B) Target B16.F10 cells were coated with mouse anti-human/mouse Trp1mAb (clone TA99) and (C) Target CT26-HER2 cells were coated with mouseanti-human HER2 mAb (clone 7.16.4). Purified NK cells were added to theantibody-coated target cells at a 5:1 and 4:1 ratio, respectively. Celldeath was determined by measuring release of Lactate Dehydrogenase (LDH)into the cell culture medium. In both assays, target cell lysis wasstronger when NK cells were activated in vivo by rMVA or rMVA-CD40Lcompared to PBS. Activation by rMVA-CD40L resulted in the highest lyticactivity. CT26-HER2 cells incubated with different concentrations ofanti-human HER2 (0.1 to 10 μg/ml anti-HER2) were also more efficientlykilled by rMVA-CD40L activated NK cells as compared to rMVA activated NKcells (shown in D).

Shown in FIG. 10E, ex vivo ADCC activity of NK cells was analyzed usingMVA-HER2-Twist-CD40L. Balb/c mice were immunized IV either with PBS orMVA-HER2-Twist-CD40L. CT26-HER2 cells were coated with 5, 0.5, and 0.05μg/ml of mouse anti-human HER2 mAb (clone 7.16.4). Purified NK cellswere added to the antibody-coated target cells at a 5:1 ratio. Celldeath was determined by measuring release of Lactate Dehydrogenase (LDH)into the cell culture medium. Target cell lysis was stronger when NKcells were activated in vivo by MVA-HER2-Twist-CD40L compared to PBS.

Thus, ADCC-mediated killing of tumor cells expressing relevant humantumor antigens can be enhanced by rMVA and especially rMVA-CD40L andMVA-HER2-Twist-CD40L mediated NK cell activation. Furthermore, rMVA andespecially rMVA-CD40L and MVA-HER2-Twist-CD40L enhanced ADCC activity isobserved over a wide dose range of antibody.

Example 6: Intravenous Administration of Recombinant MVA Induces StrongCD8 T Cell Responses

C57BL/6 mice were immunized IV or SC with 5×10⁷ TCID₅₀ MVA-OVA on days 0and 16. On days 7 and 22, OVA-specific CD8 T cell responses in the bloodwere assessed by flow cytometry after staining with H-2K^(b)/OVA₂₅₇₋₂₆₄dextramers. Shown in FIG. 11, on day 7 the frequency of OVA-specific CD8T-cells was 9-fold higher as compared to SC injections. On day 22,OVA-specific T-cells were 4-fold higher than after SC injection.

Example 7: Intravenous Administration of Recombinant MVA-CD40L FurtherEnhances CD8 T Cell Responses

Shown in FIG. 12, C57BL/6 mice were immunized intravenously with 5×10⁷TCID₅₀ MVA-OVA or MVA-OVA-CD40L on days 0 and 35. OVA-specific CD8 Tcell responses in the blood were assessed by flow cytometry afterstaining with H-2Kb/OVA₂₅₇-264 dextramers. At the peak of the primary(day 7) and secondary (day 39) response, the frequency of OVA-specificCD8 T cells was enhanced 4-fold and 2-fold, respectively afterMVA-OVA-CD40L compared to MVA-OVA immunization (Lauterbach et al. 2013).

Example 8: Prime-Boost Immunization Shows Repeated NK Cell Activationand Proliferation

TABLE 1 Examples 8-10 IV immunization scheme groups prime day 0 boostday 21 boost day 42 PBS PBS PBS PBS rMVA hom rMVA rMVA rMVA rMVA-CD40Lhom rMVA-CD40L rMVA-CD40L rMVA-CD40L rMVA-CD40L het rMVA-CD40L rMVA rMVA

C57BL/6 mice were immunized IV as shown in Table 1 (recombinant MVAdosages were at 5×10⁷ TCID₅₀). NK cells (CD3⁻ NKp46) were analyzed inthe blood by flow cytometry one and four days after second and thirdimmunization. Shown in FIGS. 13A and 13B are the GMFI CD69 (A) and thefrequency of Ki67⁺ NK cells (B). FIGS. 13A and 13B illustrate that NKcells are activated by each immunization despite the presence ofanti-vector immunity. This unexpected finding supports combination ofantibody therapy with boost immunizations that would activate NK cells.Thus, when cancer patients are treated multiple times with recombinantMVA and mount anti-vector responses, NK cell activation is not impaired.In contrast, each treatment leads to de novo NK cell activation.

Example 9: Prime-Boost Immunization Shows Stronger Induction of CD4 THelper Cells

C57BL/6 mice were immunized as shown in Table 1 (recombinant MVA dosageswere at 5×10⁷ TCID₅₀). Serum cytokine levels were quantified at 6 hourspost immunization by a multiplex bead assay (Luminex). Shown are theresults from the expression of the named cytokines. 14A) IL-6; 14B)CXCL10; 14C) IFN-α; 14D) IL-22; 14E) IFN-γ; 14F) CXCL1; 14G) CCL4; 14H)CCL7; 14I) CCL2; 14J) CCL5; 14K) TNF-α; 14L) IL-12p70; and 14M) IL-18.

Shown in FIGS. 14A-14M, rMVA-CD40L hom-treated mice had a similarcytokine profile as mice primed with rMVA and boosted with rMVA-CD40L(rMVA-CD40L het). rMVA hom-treated mice displayed lower levels of IL-6,IL12p70, IL-22, IFN-α, TNF-α, CCL2, CCL5 and CXCL1 after the first andsecond immunization compared to mice primed with rMVA-CD40L. A cytokineabsent after the prime but highly produced after second and thirdimmunization was IL-22. IL-22 is largely produced by effector T helpercells and subpopulations of innate lymphocyte cells. The higherexpression of IL-22 in rMVA-CD40L het or rMVA-CD40L hom-treated micethus indicates stronger induction CD4 T helper responses by rMVA-CD40Limmunization. Overall, IV rMVA and rMVA-CD40L immunization induced highsystemic cytokine responses that are highest in mice primed withrMVA-CD40L.

Example 10: Prime-Boost Immunization Shows Stronger Antigen Specific CD8T Cell Responses

C57BL/6 mice were immunized IV as shown in Table 1 (recombinant MVAdosages were at 5×10⁷ TCID₅₀). Similar to NK cells, induction ofantigen-specific CD8 T cell responses after repetitive immunization wasassessed. Shown in FIG. 15, CD8 T cell frequencies were drasticallyenhanced after the second and third immunization with rMVA andrMVA-CD40L. Of note, after the second immunization, about 80% of all PBLwere CD8 T cells (A). While the vector-specific (B8) CD8 T cell responsepeaked after the second immunization (B), transgene-specific (OVA)responses could be further boosted with a third immunization (C). Thisresulted in increased ratios of OVA/B8-specific CD8 T cells (D),indicating an immune focusing towards the transgene. Importantly, theseresults demonstrate that antigen-specific T cell responses can beboosted by IV immunization in the presence of anti-vector immunity.Thus, in cancer patients, multiple treatments might result in strongertumor-antigen specific CD8 T cell responses.

Example 11: Prime-Boost Immunization Shows Stronger Memory CD8 and CD4 TCell Responses

C57BL/6 mice were immunized IV as shown in Table 1. The results areshown in FIG. 16. Phenotypically, effector and effector memory T cellscan be identified by the expression of CD44 and the lack of surfaceCD62L. Monitoring CD44′ CD62L-CD8 (A) and CD4 (B) T cells in the blooddemonstrated that repeated IV immunization induces expansion of effectorand effector memory T cells. Interestingly, mice that received eitherrMVA-CD40L hom or rMVA-CD40L het had about 2.5-fold more circulatingeffector CD4 T cells than mice primed with rMVA (B, day 25). Thisindicates that systemic priming with rMVA-CD40L induces stronger CD4 Tcell responses than rMVA.

Example 12: Intravenous Administration of Recombinant MVA Results inStrong Anti-Tumor Effects in Treating Melanoma

C57BL/6 mice bearing palpable B16.OVA tumors were primed (dotted line)either IV or SC with PBS, MVA-OVA (rMVA) or MVA-OVA-CD40L (rMVA-CD40L)(recombinant MVA dosages were at 5×10⁷ TCID₅₀). At 7 and 14 days afterprime immunization, the mice received subsequent boosts with FPV-OVA at5×10⁷ TCID₅₀ (dashed lines). Tumor growth was measured at regularintervals. Shown in FIG. 17 are tumor mean volume (A) and survival oftumor-bearing mice by day 45 after tumor inoculation (B). Thus, primingof B16.OVA tumor bearing mice IV with rMVA-CD40L provides a strongeranti-tumor effect as compared to both SC rMVA-CD40L or SC or IV rMVA.

Example 13: A Single Intravenous Administration of Recombinant MVAResults in Strong Anti-Tumor Effects

C57BL/6 mice bearing palpable B16.OVA tumors were IV vaccinated as shownin Table 2. Tumor growth was measured at regular intervals. Shown inFIG. 18 is tumor mean volume. The results indicate that a singletherapeutic immunization with rMVA-CD40L is as strong as homologous orheterologous prime/boost immunizations. Importantly, these datahighlight the potent anti-tumor activity of rMVA-CD40L.

TABLE 2 Vaccination scheme corresponding to Example 13 Group Day 8 PrimeDay 15 Boost PBS PBS none rMVA rMVA none rMVA 2× rMVA rMVA rMVA →MVA-CD40L rMVA rMVA-CD40L rMVA → CD40L rMVA-CD40L none rMVA-CD40L → rMVArMVA-CD40L rMVA rMVA-CD40L 2× rMVA-CD40L rMVA-CD40L rMVA-CD40L → rFPVrMVA-CD40L rFPV

Example 14: CD8 T-Cells are Important for rMVA-CD40L Mediated TumorControl

C57BL/6 mice bearing palpable B16.OVA tumors were immunized viaintravenous administration with PBS, MVA-OVA (rMVA) or MVA-OVA-CD40L(rMVA-CD40L) (recombinant MVA dosages were at 5×10⁷ TCID₅₀). Micereceived 5 intraperitoneal injections of 200 μg anti-CD8 antibody whereindicated. Shown in FIG. 19, immunization with rMVA-CD40L inducedstronger overall CD8 T cell responses (A) as well as strongerneo-antigen (OVA)-specific CD8 T cell responses (B) compared to rMVA. C)represents overall survival. These data indicate that rMVA-CD40L inducessuperior CD8 T cell responses in a tumor setting and that the inducedCD8 T cell responses 10 are important for the anti-tumor effect seenafter therapeutic immunization.

Example 15: IV Administration of Recombinant MVA Encoding at Least TwoTAAs is More Effective

C57BL/6 mice bearing palpable B16.OVA tumors were immunized IV eitherwith PBS, MVA-OVA-TRP2, MVA-OVA-TRP2-CD40L or MVA-OVA-CD40L.

Shown in FIG. 20, tumor growth was measured at regular intervals and isdisplayed as mean diameter of individual mice (A) and mean volume (B).Survival is shown in (C). These data indicate that a single therapeuticimmunization with rMVA-CD40L encoding two TAAs is more efficient thanimmunization with rMVA-CD40L encoding only one TAA.

Example 16: Intravenous Administration of Recombinant MVA-CD40LIncreased T-Cell Infiltration in the Tumor Microenvironment

C57BL/6 mice bearing palpable B16.OVA tumors were immunizedintravenously with PBS, rMVA (MVA-OVA) or rMVA-CD40L (MVA-OVA-CD40L)(recombinant MVA dosages were at 5×10⁷ TCID₅₀). After 7 days, mice weresacrificed. As shown in FIGS. 21A and 21 B, the frequency anddistribution of CD8⁺ T cells and OVA₂₅₇₋₂₆₄-specific CD8+ T cells wasanalyzed among leukocytes in spleen, tumor-draining lymph nodes (TDLN)and tumor tissues. In FIG. 21C, geometric mean fluorescence intensity(GMFI) of PD-1 and Lag3 on tumor-infiltrating OVA₂₅₇₋₂₆₄-specific CD8⁺ Tcells was analyzed; In FIG. 21D, representative dot plots oftumor-infiltrating CD8′ T cells of Ki67 and PD-1 expression is shown. InFIG. 21E, frequencies of tumor-infiltrating Ki67⁺ CD8⁺ T cells and GMFIof PD-1 are shown; in FIG. 21F, frequency of tumor-infiltratingregulatory T cells (Treg) among leukocytes is illustrated. In FIG. 21G,frequencies of PD-1^(high)- and PD-1^(neg)-tumor-infiltrating Treg areshown. Taken together, these data show that rMVA-CD40L immunizationleads to a more pronounced infiltration of TAA-specific CD8 T cells intothe tumor microenvironment (TME) and that these CD8 T cells expresslower amounts of markers associated with ‘immune exhaustion’ compared toPBS and rMVA immunization. Furthermore, rMVA-CD40L immunization leads toa reduction of highly suppressive Treg in the TME compared to PBS.

Example 17: Intravenous Administration of Recombinant MVA-CD40LIncreased Longevity of T-Cell Infiltration of Tumor Microenvironment

TCR-transgenic OVA-specific CD8 T cells (OT-I) were intravenouslytransferred into B16.OVA tumor bearers when tumors were palpable. Whentumors reached at least 60 mm³ in volume animals were immunized withMVA-BN, MVA-OVA (rMVA), or MVA-OVA-CD40L (rMVA-CD40L) (recombinant MVAdosages were at 5×10⁷ TCID₅₀). After 17 days, mice were sacrificed andanalyzed for A) Frequency of CD8⁺ T cells among leukocytes in tumortissues; B) Frequency of Lag3⁺ PD1⁺ within CD8⁺ T cells; C) Frequency ofEomes+PD1⁺ T cells within CD8⁺ T cells; D) Presence of OT-I-transgenicCD8⁺ T cells within the TME upon immunization; and E) Frequency of Lag3⁺PD1⁺ exhausted T cells within OT-I+CD8⁺ T cells; and F) Frequency ofEomes+PD1⁺ exhausted T cells within OT-I+CD8⁺ T cells. The results areshown in FIG. 22A-22D. These data indicate that TAA-specific CD8 T cellsthat are recruited into the TME upon rMVA-CD40L immunization show lesssigns of immune exhaustion than after control treatment (MVA-BN withoutencoded TAA) or rMVA immunization even after prolonged exposure to theTME.

Example 18: Intravenous Administration of Recombinant MVA-CD40LDecreased Levels of Tree in Tumor Microenvironment

Purified OVA-specific TCR-transgenic CD8 T cells (OT-I) wereintravenously transferred into B16.OVA tumor bearers when tumors werepalpable. When tumors reached at least 60 mm³ in volume animals wereimmunized with MVA-BN, MVA-OVA (rMVA), or MVA-OVA-CD40L (rMVA-CD40L)(recombinant MVA dosages were at 5×10⁷ TCID₅₀). After 17 days mice weresacrificed and analyzed for: A) Frequency of Foxp3⁺CD4⁺ Treg among CD4⁺T cells in tumor tissues; and B) Ratio of CD8⁺ CD44⁺ effector T cells toFoxp3⁺ CD4⁺ Tregs. The results are shown in FIG. 23. Taken together,these data show that even after prolonged exposure to the TME, a singleimmunization with rMVA-CD40L leads to an increased Teff/Treg ratiocompared control treatment (MVA-BN without encoded TAA) or rMVAimmunization.

Example 19: MVA-OVA-CD40L Safer for Humans as Compared to AgonisticCD40mAb

TABLE 3 Immunization schedule Example 19 groups prime day 0 boost day 21boost day 42 PBS PBS PBS PBS rMVA hom rMVA rMVA rMVA rMVA-CD40L homrMVA-CD40L rMVA-CD40L rMVA-CD40L rMVA-CD40L het rMVA-CD40L rMVA rMVAαCD40 Anti-CD40 Anti-CD40 Anti-CD40 mAb mAb mAb

C57BL/6 mice were injected IV either with PBS, MVA-OVA (rMVA),MVA-OVA-CD40L (rMVA-CD40L) or anti-CD40 (FGK4.5) as shown in Table 3(recombinant MVA dosages were at 5×10⁷ TCID₅₀, anti-CD40 dosage was at100 μg). Serum concentration of alanine aminotransferase (ALT) wasquantified one day after each immunization by ELISA. Shown in FIG. 24,ALT levels were highest after the first injection of anti-CD40. The ALTconcentration after the first immunization with rMVA-CD40L was 2-foldlower than after anti-CD40 treatment. Importantly, serum ALT levelsafter repeated rMVA and rMVA-CD40L immunization were not enhanced.Although repeated injection of anti-CD40 antibody also did not result inenhanced ALT levels, 3 of 4 mice died in this group (FD=found dead).Also shown in FIG. 21 lower serum levels of alanine aminotransferase(ALT) were seen after rMVA and rMVA-CD40L immunization compared toanti-CD40 immunoglobulin injection. Thus, in contrast to the describedliver toxicity of agonistic anti-CD40 (Medina-Echeverz et al., 2015), nosuch toxicity was observed after repeated IV injection of rMVA andrMVA-CD40L.

Example 20: Increased Overall Survival and Tumor Reduction in IVAdministration of rMVA-CD40L

Balb/c mice bearing palpable CT26.HER2 tumors were immunized IV withMVA-AH1A5-p15-TRP2-CD40L (rMVA-CD40L) or received PBS. Tumor growth wasmeasured at regular intervals. Shown in FIG. 25 are the tumor meanvolume (A) and survival (B). These data indicate that a singletherapeutic immunization with rMVA-CD40L is efficiently prolongingsurvival in a colon cancer model.

Example 21: Increased Overall Survival and Tumor Reduction in IVAdministration of rMVA-CD40L+Anti-TrP1

C57BL/6 mice bearing palpable B16.OVA tumors (at least 60 mm in volume)were immunized intravenously with MVA-OVA-CD40L (mBNbc115) at 5×10⁷TCID₅₀ on day 8. Mice were intraperitoneally injected with 200 μganti-Trp1 (clone TA99) twice/week starting on day 5. Results are shownin FIGS. 26A and 26B for tumor mean volume and overall survival rate,respectively. Shown in the Figures, the combination of rMVA-CD40L andthe anti-Trp1 mAb showed an overall reduction in tumor volume andincrease in overall survival rate as compared to the anti-Trp1 mAb andthe rMVA-CD40L by themselves. Thus, combination of a tumor-specificantibody and rMVA-CD40L administered intravenously has a significantlyincreased anti-tumor activity as compared to either a non-IVadministration of rMVA-CD40L or an administration of antibody alone.

Example 22: Construction of Recombinant MVA Viruses MVA-mBN445,MVA-mBN451, MVA-mBNbc197, MVA-mBNbc195, MVA-mBNbc388, MVA-mBN bc389, andMVA-mBN484

Generation of recombinant MVA viruses that embody elements of thecombination therapy (e.g., MVA-mBN445, MVA-mBN451 and MVA-mBN484) wasdone by insertion of the indicated transgenes with their promoters intothe vector MVA-BN. Transgenes were inserted using recombination plasmidscontaining the transgenes and a selection cassette, as well as sequenceshomologous to the targeted loci within MVA-BN. Homologous recombinationbetween the viral genome and the recombination plasmid was achieved bytransfection of the recombination plasmid into MVA-BN infected CEFcells. The selection cassette was then deleted during a second step withhelp of a plasmid expressing CRE-recombinase, which specifically targetsloxP sites flanking the selection cassette, therefore excising theintervening sequence.

For construction of MVA-BN mBNbc197 and MVA-BN mBNbc195, recombinationplasmids were used for the two or three transgenes for mBNbc197(MVA-OVA-TRP2) and mBNbc195 (MVA-OVA-TRP2-CD40L), respectively. Theplasmids included insert sequences which are also present in MVA.Nucleotide sequences encoding the OVA and TRP2 and/or CD40L antigenswere present between the MVA insert sequences to allow for recombinationinto the MVA viral genome. Thus, a plasmid was constructed for eachconstruct that contained the OVA and TRP2 and/or CD40L coding sequences,each downstream of a promoter.

For the construction of mBN451, the recombination plasmid included twotransgenes HER2v1 and Brachyury (SEQ ID NO: 1 and SEQ ID NO: 3,respectively), each preceded by a promoter sequence, as well assequences which are identical to the targeted insertion site withinMVA-BN to allow for homologous recombination into the viral genome. TheHER2 and Brachyury coding sequences (or nucleotide sequences) are SEQ IDNO: 2 and SEQ ID NO: 4, respectively.

For the construction of mBN445 the recombination plasmid included thethree transgenes HER2v1, Brachyury, and CD40L (SEQ ID NO: 1, SEQ ID NO:3, and SEQ ID NO: 11, respectively), each preceded by a promotersequence, as well as sequences which are identical to the targetedinsertion site within MVA-BN to allow for homologous recombination intothe viral genome. The HER2, Brachyury, and CD40L coding sequences (ornucleotide sequences) are SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 12,respectively.

For construction of mBNbc388 the recombination plasmid included thethree transgenes HER2v1, Twist, and CD40L (SEQ ID NO: 1, SEQ ID NO: 15,and SEQ ID NO: 17, respectively), each preceded by a promoter sequence,as well as sequences which are identical to the targeted insertion sitewithin MVA-BN to allow for homologous recombination into the viralgenome. The HER2v1, Twist, and CD40L coding sequences (or nucleotidesequences) are SEQ ID NO: 2, SEQ ID NO: 16, and SEQ ID NO: 18,respectively.

For construction of mBNbc389 the recombination plasmid included the twotransgenes HER2v1 and Twist (SEQ ID NO: 1, SEQ ID NO: 15, respectively),each preceded by a promoter sequence, as well as sequences which areidentical to the targeted insertion site within MVA-BN to allow forhomologous recombination into the viral genome. The HER2, Twist, andCD40L coding sequences (or nucleotide sequences) are SEQ ID NO: 2 andSEQ ID NO: 16 respectively.

For construction of mBN484 the recombination plasmid included the threetransgenes HER2 v.2, Brachyury, and CD40L (SEQ ID NO: 13, SEQ ID NO: 3,and SEQ ID NO: 11, respectively), each preceded by a promoter sequence,as well as sequences which are identical to the targeted insertion sitewithin MVA-BN to allow for homologous recombination into the viralgenome. The HER2 v.2, Twist, and CD40L coding sequences (or nucleotidesequences) are SEQ ID NO: 14, SEQ ID NO: 4, and SEQ ID NO: 12,respectively.

For generation of the above described mBN MVAs, (e.g, .mBN445, mBN451,and mBN484), CEF cell cultures were each inoculated with MVA-BN andtransfected each with the corresponding recombination plasmid. In turn,samples from these cell cultures were inoculated into CEF cultures inmedium containing drugs inducing selective pressure, andfluorescence-expressing viral clones were isolated by plaquepurification. Loss of the fluorescent protein-containing selectioncassette from these viral clones was mediated in a second step byCRE-mediated recombination involving two loxP sites flanking theselection cassette in each construct. After the second recombinationstep only the transgene sequences (e.g., HER2, Brachyury, and/or CD40L)with their promoters inserted in the targeted loci of MVA-BN wereretained. Stocks of plaque-purified virus lacking the selection cassettewere prepared.

Expression of the identified transgenes was subsequently demonstrated incells inoculated with the described construct (See e.g., FIG. 27).

Generation of MVA-mBNbc197, mBNbc195, mBNbc388, and mBNbc389 was carriedout by using a cloned version of MVA-BN in a bacterial artificialchromosome (BAC). Recombination plasmids containing the differenttransgenes for mBNbc197 (MVA-Ova-Trp2), mBNbcl95 (MVA-Ova-Trp2-CD40L),mBNbc388 and mBNbc389 were used. The plasmids included sequences thatare also present in MVA and therefore allow for specific targeting ofthe integration site. Nucleotide sequences encoding the OVA, Her2 v1,TwistTrp2 and/or CD40L antigens were present between the MVA sequencesthat allow for recombination into the MVA viral genome. Thus, a plasmidwas constructed for each construct that contained the OVA, Her2 v1,Twist, Trp2 and/or CD40L coding sequences, each downstream of apromoter. Briefly, infectious viruses were reconstituted from BACs bytransfecting BAC DNA into BHK-21 cells and superinfecting them withShope fibroma virus as a helper virus. After three additional passageson CEF cell cultures, helper-virus free MVA-mBNbcl97, MVA-mBNbcl95,MVA-mBNbc388 and MVA-mBNbc389 were obtained. An exemplary MVA generationis also found in Baur et al. ((2010) J. Virol. 84: 8743-52).

Example 23: Heterologous Expression of MVA-Her2-Brachyury-CD40L

HeLa cells were left untreated (mock) or infected with MVA-BN orMVA-HER2-Brachyury-CD40L (MVA-mBN445). After overnight culture, cellswere stained with anti-HER2-APC (clone 24D2), anti-Brachyury (rabbitpolyclonal)+anti-rabbit IgG-PE and anti-CD40L-APC (clone TRAP1). Shownin FIG. 27A-27D, flow cytometric analysis revealed expression of allthree transgenes.

Example 24: Synthetic HER2 Proteins Prevent Binding of Trastuzumab andPertuzumab

Mouse colon carcinoma CT26 cells were infected withMVA-HER2-Brachyury-CD40L (MVA-mBN484) at an MOI of 1. 24 hours later,cells were incubated with 5 μg/ml of the HER2 antibodies Trastuzumab,Pertuzumab or 24D2. Figure shows expression levels of HER2 normalized toclone 24D2 HER2 staining. Data expressed as Mean±SEM, representative oftwo independent experiments. The results are shown in FIG. 28.

Example 25: Enhanced Activation of Human DCs by MVA-HER2-Brachyury-CD40L

Monocyte-derived dendritic cells (DCs) were generated after enrichmentof CD14′ monocytes from human PBMCs and cultured for 7 days in thepresence of GM-CSF and IL-4 according to protocol (Miltenyi, MO-DCgeneration tool box). DCs were stimulated with MVA-HER2-Brachyury orMVA-HER2-Brachyury-CD40L. Shown in FIG. 29 expression of A) CD40L, B)CD86, and C) and MHC class II was analyzed by flow cytometry. Shown inD), the concentration of IL-12p70 in the supernatant was quantified byluminex after over-night culture.

This experiment demonstrates that rMVA-HER2-Brachyury-CD40L stimulateshuman DCs, inducing their activation and thus enhancing their capabilityto present antigens. The production of the Th1 polarizing and NK cellactivating cytokine IL-12p70 by stimulated human DCs indicates thatMVA-HER2-Brachyury-CD40L activates human DCs towards a pro-inflammatoryphenotype.

Example 26: Increase in Overall Survival and Tumor Reduction inIntravenous Administration of mBNbc388 and mBNbc389

Mouse Colon Carcinoma MC38 cells expressing HER2 (MC38.HER2) were s.c.(subcutaneously) injected into C57BL/6 mice. Mouse Colon Carcinoma CT26cells expressing HER2 (CT26.HER2) were s.c. injected into Balb/c mice.In both mice groups, when tumors were above 50 mm³ mice bearingMC38.HER2 tumors (FIGS. 30A and 30B) and CT26.HER2 tumors (FIG. 31A andFIG. 31B) were immunized (dotted line) either with PBS, MVA-CD40L orMVA-HER2-Twist-CD40L IV. Tumor growth was measured at regular intervals.In A) tumor mean volume was measure and in B) overall survival oftumor-bearing mice by day 60 after tumor inoculation.

Because the mouse homolog of Brachyury is neither highly expressed innormal mouse tissues nor predominantly expressed in mouse tumor tissues,the efficacy of Brachyury as a target for an active immunotherapy cannotbe studied effectively in a mouse model system (see WO 2014/043535,which is incorporated by reference herein). Twist, the mouse homolog ofthe Human Brachyury is used in mouse models is a predictive model forBrachyury function in humans. This was demonstrated in WO 2014/043535.Like Brachyury, the mouse homolog of the EMT regulator Twist bothpromotes the EMT during development by down-regulatingE-cadherin-mediated cell-cell adhesion and up-regulating mesenchymalmarkers and is predominantly expressed in mouse tumor tissue (see, e.g.,FIG. 5 and Example 8 of WO 2014/043535). Therefore, the study of aTwist-specific cancer vaccine in mice is very likely to have strongpredictive value regarding the efficacy of a Brachyury-specific cancervaccine in humans (Id.).

Example 27: Intravenous Administration of MVA-HER2-Twist-CD40L(mBNbc388) Enhances Infiltration of HER2 Specific CD8+ T Cells intoTumors

Balb/c mice bearing CT26.HER2 tumors received intravenously either PBSor 5×10⁷ TCID₅₀ MVA-HER2-Twist-CD40L. Seven days later, mice weresacrificed, spleen and tumor-infiltrating CD8⁺ T cells isolated bymagnetic cell sorting and cultured in the presence of HER2peptide-loaded dendritic cells for 5 hours. Graph shows percentage ofCD44⁺ IFNγ⁺ cells among CD8⁺ T cells. Results are shown as Mean±SEM. Theresults, illustrated in FIG. 32A, demonstrate that the variousembodiments of the present invention are tumor specific.

Example 28: Increase in Overall Survival and Tumor Reduction inIntravenous Administration of mBNbc388 and mBNbc389 in combination withTrastuzumab

Balb/c mice bearing CT26.HER2 tumors above 100 mm³ mean volume received5 μg of either human IgG1 or Trastuzumab intraperitoneally on day 15after tumor inoculation. 2 days after first injection of human IgG1 orTrastuzumab, mice received intravenously PBS or 5×10⁷ TCID₅₀ ofMVA-HER2-Twist-CD40L as illustrated in FIG. 31. In this example, HumanIgG1 is used as an experimental control for Trastuzumab, since IgG1 doesnot bind HER2 as Trastuzumab does. Tumor growth and overall survivaltime was measured at regular intervals. (A) Tumor mean volume and (B)mouse survival over time.

Shown in FIGS. 33 (A) and (B), the combination of MVA-HER2-Twist-CD40Lwith Trastuzumab was particularly effective in reducing larger tumorsand extending the overall survival rate of mice with those large tumorseven at reduced dosage of antibody as compared to Example 21.

Example 29: Overall Survival and Tumor Reduction in IV Administration ofMVA-HER2-Twist-CD40L in Orthotopic Breast Cancer Model

Balb/c mice bearing orthotopic 4T1.HER2 tumors are primed IV with 5×10⁷TCID₅₀ MVA-HER2-Twist (mBNbc389) and/or MVA-HER2-Twist-CD40L (mBNbc388).Tumor growth will be measured at regular intervals and overall survivalwill be recorded.

Example 30: Strong NK Cell Mediated Toxicity from an IV Administrationof MVA-HER2-Twist-CD40L+Anti-HER2

C57BL/6 mice are immunized IV with 5×10⁷ TCID₅₀ MVA-HER2-Twist,MVA-HER2-Twist plus anti-Her2, or PBS. 24 hours later mice aresacrificed and splenic NK cells are purified by magnetic cells sortingand will be used as effectors in an effector: target killing assay.Briefly NK cells are cultured with CFSE-labelled MCH class I-deficientYAC-1 cells at the ratios shown in Example 4. Specific killing isassessed by quantifying unviable CFSE+ YAC-1 cells by flow cytometry.

Example 31: IV Administration of MVA-HER2-Twist-CD40L+Anti-HER2 EnhancesADCC

C57BL/6 mice are treated IV either with 25 μg anti-CD4,MVA-HER2-TWIST-CD40L+5 μg rat IgG2b, or 1 μg anti-CD4 orMVA-HER2-TWIST-CD40L+1 μg anti-CD4 24 hours later mice are sacrificedand CD4 T cell (CD3⁺CD4⁺) depletion in the liver is analyzed by flowcytometry. 25 μg anti-CD4 was defined as 100% specific killing.

To assess ex vivo ADCC activity of NK cells, C57BL/6 (B) or Balb/c (C)mice are immunized IV either with PBS, MVA-HER2-TWIST orMVA-HER2-TWIST-CD40L. 24 hours later mice are sacrificed; splenic NKcells are purified by magnetic cell sorting and used as effectors inantibody-dependent effector: target killing assays. (B) Target MC38-HER2cells are coated with mouse anti-human/mouse HER2 mAb. Target CT26-HER2cells are coated with mouse anti-human HER2 mAb. Purified NK cells areadded to the antibody-coated target cells at a 5:1 and 4:1 ratio,respectively. Cell death is determined by measuring release of LactateDehydrogenase (LDH) into the cell culture medium.

Example 32: Increased Overall Survival and Tumor Reduction in IVAdministration of rMVA-CD40+Anti-HER2 at Decreasing Concentrations ofHER2 Antibody

Balb/c mice bearing palpable CT26-HER2 tumors are immunized IV withMVA-HER2-Twist-CD40L at 5×10⁷ TCID₅₀ on day 8. Mice wereintraperitoneally injected with decreasing dosages of anti-HER2 (200 μg,100 μg, 25 μg) twice/week starting on day 5. Tumor mean volume andoverall survival rate are analyzed as compared to mice injected withjust 200 μg anti-HER2 antibody and mice injected with justMVA-HER2-TWIST-CD40L.

Example 33: Increased Overall Survival and Tumor Reduction in IVAdministration of rMVA-CD40L+Anti-CD52 Antibody

Balb/c mice bearing CT26.CD52 tumors receive 5 μg of either human IgG1or anti-CD52 (alemtuzumab) intraperitoneally after tumor inoculation.After first injection of human IgG1 or alemtuzumab, mice receiveintravenously PBS or 5×10⁷ TCID₅₀ of MVA-CD52-CD40L. Tumor growth andoverall survival time are measured at regular intervals. (A) Tumor meanvolume and (B) mouse survival over time.

Example 34: Increased Overall Survival and Tumor Reduction in IVAdministration of rMVA-CD40L+Anti-EGFR Antibody

Balb/c mice bearing CT26.EGFR tumors receive 5 μg of either human IgG1or anti-EGFR (cetuximab) intraperitoneally after tumor inoculation.After first injection of human IgG1 or cetuximab, mice receiveintravenously PBS or 5×10⁷ TCID₅₀ of MVA-EGFR-CD40L. Tumor growth andoverall survival time are measured at regular intervals. (A) Tumor meanvolume and (B) mouse survival over time.

It will be apparent that the precise details of the methods orcompositions described herein may be varied or modified withoutdeparting from the spirit of the described invention. We claim all suchmodifications and variations that fall within the scope and spirit ofthe claims below.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and either one letter code or three letter code for amino acids,as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acidsequence is shown, but the complementary strand is understood asincluded by any reference to the displayed strand.

Synthetic Her2 v1 amino acid sequence (1,145 amino acids): SEQ ID NO: 1MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTRTFKSMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPAANQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPAADQCVACAHYKDPPACVARCPSGVKPDLSYMPIWAFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTSIISAVVGILLVVVLGVVFGILIKRRQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIMVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLGPASPLDSTFYRSLLEDDDMGDLVDAEEALVPQQGFFCPDPAPGAGGMVHHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMGAAKGLQSLPTHDPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPELGLDVPVSynthetic Her2 v1 nucleotide sequence (3441 nucleotides): SEQ ID NO: 2atggaactggctgctctgtgtagatggggactgctgcttgctctgttgcctcctggagctgcttctacccaagtgtgcacaggcaccgacatgaagctgagactgcctgcttctcctgagacacacctggacatgctgagacacctgtaccagggatgtcaggtggtgcagggaaatctggaactgacctacctgcctaccaacgccagcctgagctttctgcaggacatccaagaggtgcagggatacgtgctgatcgctcacaatcaagtgagacaggtgccactgcagaggctgagaatcgttagaggcacccagctgttcgaggacaactatgctctggctgtgctggacaatggcgaccctctgaacaacaccacacctgtgacaggagcttctcctggtggactgagagaactgcagctgagaagcctgaccgagatcctgaaaggaggagtgctgatccagcggaaccctcagctgtgctaccaggacaccatcctgtggaaggacatcttccacaagaacaaccagctggctctgacactgatcgacaccaacagaagcagagcctgccatccttgctctcccatgtgcaagggctctagatgttggggagagagcagcgaggattgccagagcctgaccagaacagtgtgtgctggaggatgtgccagatgcaaaggacctctgcctaccgactgctgccacgagcaatgtgcagotggatgtacaggaccaaagcactctgattgcctggcctgcctgcacttcaaccactctggaatctgcgagctgcactgtcctgctctggtcacctacaacacacggaccttcaagagcatgcctaatcctgaaggcagatacacctttggagccagctgtgtgacagcctgtccttacaactacctgagcaccgacgtgggcagctgcacactcgtttgtcctgctgccaatcaagaagtgacagccgaggacggcacccagagatgcgagaagtgtagcaagccttgcgctagagtgtgttacggactcggcatggaacacctgagagaagtgagagccgtgaccagtgccaacatccaagagtttgctggctgcaagaagatattggcagcctcgccttcctgcctgagagcttcgatggcgatcctgccagcaatactgctcctctgcagcctgaacagctccaggtgttcgagacactggaagagatcacaggctacctgtacatcagcgcatggccagacagectgcctgacctgtccgtgttccagaacctgcaagtgatcagaggcagaatcctgcacaacggagcctattctctgaccctgcaaggcctgggaatcagctggctgggactgagatccctgagagagottggatctggcctggctctgatccaccacaatacccacctgtgcttcgtgcacaccgtgccttgggaccagetgtttcggaatcctcatcaggctctgctgcacacagccaacagacctgaggatgagtgtgttggcgaaggcctggcttgtcaccagctctgtgctagaggacactgttggggacctggacctacacagtgtgtgaactgtagccagttcctgagaggccaagaatgcgtggaagagtgtagagttctgcagggactgcctcgcgagtacgtgaacgctagacactgtctgccttgtcatcccgagtgccagcctcagaatggcagcgtgacatgttttggaccagctgccgatcagtgcgtggcctgtgctcactataaggaccotccagcctgcgtggccagatgtcctagcggagtgaagcctgacctgagctacatgcccatctgggcatttccagatgaggaaggagcttgccagccttgtcctatcaactgcacccacagctgcgtggacctggacgataagggatgtccagccgagcagagagcctctccactgacctctatcatctctgccgtcgtgggcatcctgctggtggtggttctgggagttgtgttcggcatcctgatcaagagacggcagcagaagatccggaagtacaccatgcggagactgctgcaagagactgagctggtggaacctctgacacccagcggagctatgcctaaccaggctcagatgcggattctgaaagaaaccgagctgcggaaagtgaaggtgctcggctctggagcctttggcacagtgtacaaaggcatctggatccctgacggagagaacgtgaagattcctgtggccatcatggtgctgagagagaacacaagtcccaaggccaacaaagagatectggacgaggcctacgtgatggctggtgttggcagcccttatgtgtctagactgctgggcatctgtctgaccagcaccgtgcagctggtcactcagctgatgccttacggctuctgctggatcacgtgagagagaatagaggcagactgggctotcaggacctgctgaactggtgcatgcagatcgccaagggcatgagctacctcgaggatgtgagactggtccacagagatctggctgccagaaacgtgctcgtgaagtctcctaaccacgtgaagatcaccgacttcggactggctaggctgctggatatcgacgagacagagtaccacgctgatggaggcaaggtgcccatcaagtggatggctctggaatccatcctgagacggagattcacccaccagtccgatgtgtggtcttacggagtgacagtgtgggagctgatgaccttcggagccaagccttacgacggcatccctgccagagagatcccagatctgctggaaaagggagagagactgcctcagcctactatctgcaccatcgacgtgtacatgattatggtcaagtgttggatgatcgacagegagtgcagacccagattcagagaactggtgtccgagttctctcggatggccagagatcctcagagattcgtggtcatccagaacgaggatctgggacctgccagccctctggacagcaccttctacagatccctgctggaagatgacgacatgggtgacctggtggacgctgaagaagctctggttcctcagcagggcttcttctgccctgatcctgctccaggagcaggtggaatggtgcatcacagacacagaagctccagcaccagaagcggaggcggagatctgacactgggactcgagccatctgaggaagaggctcctagatctcctctggctecttctgaaggagctggaagcgacgttttcgacggagatcttggaatgggagctgccaaaggactccagtctctgcccacacacgacccatctccactgcagagatacagcgaggaccctaccgtgcctctgccaagcgagacagatggatatgtggcacctctgacctgctctcctcagccagaactgggacttgatgtgcctgtttgatgaSynthetic Brachyury amino acid sequence (427 amino acids nucleotides):SEQ ID NO: 3 MSSPGTESAGKSLQYRVDHLLSAVENELQAGSEKGDPTERELRVGLEESELWLRFKELTNEMIVTKNGRRMFPVLKVNVSGLDPNAMYSFLLDFVAADNHRWKYVNGEWVPGGKPEPQAPSCVYIHPDSPNFGAHWMKAPVSFSKVKLTNKLNGGGQIMLNSLHKYEPRIHIVRVGGPQRMITSHCFPETQFIAVTAYQNEEITALKIKYNPFAKAFLDAKERSDHKEMMEEPGDSQQPGYSQWGWLLPGTSTLCPPANPHPQFGGALSLPSTHSCDRYPTLRSHYAHRNNSPTYSDNSPACLSMLQSHDNWSSLGMPAHPSMLPVSHNASPPTSSSQYPSLWSVSNGAVTPGSQAAAVSNGLGAQFFRGSPAHYTPLTHPVSAPSSSGSPLYEGAAAATDIVDSQYDAAAQGRLIASWTPVSPPSMSynthetic Brachyury nucleotide sequence (1,287 nucleotides):SEQ ID NO: 4atgtctagccctggcacagagtctgctggcaagagcctccagtacagagtggaccatctgctgagcgctgtggagaatgaactgcaggctggaagcgagaagggagatcctacagaaagagagctgagagtcggactggaagagtccgagctgtggctgcggttcaaagaactgaccaacgagatgatcgtgaccaagaacggcagacggatgttccctgtgctgaaagtgaacgtgtccggactggaccctaacgccatgtacagattctgctggatttcgtggcagctgacaaccacagatggaagtacgtgaacggagagtgggtgccaggaggaaaacctgaacctcaggctectagctgcgtgtacattcaccctgacagccctaactteggagcccactggatgaaggctcctgtgtccttcagcaaagtgaagctgaccaacaagctgaacggaggaggccagatcatgctgaacagcctgcacaagtatgagcctaggatccacatcgtcagagttggaggccctcagcggatgatcaccagccactgtttccctgagacacagttcatcgcagtgaccgcttaccagaacgaggaaatcacagccctgaagatcaagtacaatcccttcgccaaggccttcctggacgccaaagagcggagcgaccacaaagaaatgatggaagaacctggcgacagccagcagcctggctattctcaatggggatggctgctgccaggcacctccacattgtgccctccagccaatcctcatcctcagtttggcggagccctgagcctgcctagcacacacagctgcgacagataccctacactgagaagccactacgctcacagaaacaacagccctacctacagcgacaatagccctgcctgtctgagcatgctgcagtcccacgacaattggtccagcctgggaatgcctgctcacccttctatgctgcctgtctctcacaacgcctctccacctacaagcagctctcagtaccctagcctttggagcgtgtccaatggagctgtgacacctggatctcaggctgccgctgtgtctaatggactgggagcccagttcttcagaggcagccctgctcactacacacctctgacacatccagtgtctgctcctagcagcagcggaagccctctctatgaaggagccgctgcagccaccgacatcgtggattctcagtatgatgctgccgcacagggcagactgatcgcctcttggacacctgtgagcccaccttccatgtgatga Brachyury protein Isoform 1 from GenBank Accession No. O15178.1(435 aa) SEQ ID NO: 5MSSPGTESAGKSLQYRVDHLLSAVENELQAGSEKGDPTERELRVGLEESELWLRFKELTNEMIVTKNGRRMFPVLKVNVSGLDPNAMYSFLLDFVAADNHRWKYVNGEWVPGGKPEPQAPSCVYIHPDSPNFGAHWMKAPVSFSKVKLTNKLNGGGQIMLNSLHKYEPRIHIVRVGGPQRMITSHCFPETQFIAVTAYQNEEITALKIKYNPFAKAFLDAKERSDHKEMMEEPGDSQQPGYSQWGWLLPGTSTLCPPANPHPQFGGALSLPSTHSCDRYPTLRSHRSSPYPSPYAHRNNSPTYSDNSPACLSMLQSHDNWSSLGMPAHPSMLPVSHNASPPTSSSQYPSLWSVSNGAVTPGSQAAAVSNGLGAQFFRGSPAHYTPLTHPVSAPSSSGSPLYEGAAAATDIVDSQYDAAAQGRLIASWTPVSPPSMCoding sequence for Brachyury protein Isoform 1 GenBank Accession No. O15178.1(1308 nt) SEQ ID NO: 6atgagcagccctggcacagagagcgccggcaagagcctgcagtaccgggtggaccatctgctgagcgccgtggagaatgagctgcaggccggctccgagaagggcgaccccaccgagagggaactgagagtgggcctggaagagtccgagctgtggctgcggttcaaagaactgaccaacgagatgatcgtgaccaagaacggcagacggatgttccccgtgctgaaagtgaacgtgtccggcctggaccccaacgccatgtacagctttctgctggacttcgtggccgccgacaaccacaggtggaaatacgtgaacggcgagtgggtgccaggcggcaaacctgagcctcaggcccccagctgcgtgtacatccaccccgacagccccaatttcggcgcccactggatgaaggcccccgtgtccttcagcaaagtgaagctgaccaacaagctgaacggcggaggccagatcatgctgaacagcctgcacaagtacgagccccggatccacattgtgcgcgtgggcggaccccagagaatgatcaccagccactgcttccccgagacacagtttatcgccgtgaccgcctaccagaacgaggaaatcaccgccctgaagatcaagtacaaccccttcgccaaggccttcctggacgccaaagagcggagcgaccacaaagaaatgatggaagaacccggcgacagccagcagcctggctacagccagtggggctggctgctgccaggcacctccactctgtgcccccctgccaaccctcaccctcagttcggcggagccctgagcctgcctagcacacacagctgcgacagataccccaccctgcggagccacagaagcagcccctaccccagcccatacgcccaccggaacaacagccccacctacagcgacaactcccccgcctgcctgagcatgctgcagagccacgacaactggtccagcctgggcatgcctgcccaccctagcatgctgcccgtgtcccacaatgccagcccccctaccagcagctcccagtaccctagcctgtggagcgtgtccaatggcgccgtgacacctggatctcaggccgctgccgtgagcaatggcctgggagcccagttctttagaggcagccctgcccactacacccctctgacccaccctgtgtccgcccctagctccagcggcagccctctgtatgaaggcgccgctgcagccaccgatatcgtggacagccagtacgatgccgccgctcagggcagactgatcgccagctggacccccgtgtctccccccagcatgtgaBrachyury protein Isoform 1 (L254V). (435 aa) SEQ ID NO: 7MSSPGTESAGKSLQYRVDHLLSAVENELQAGSEKGDPTERELRVGLEESELWLRFKELTNEMIVTKNGRRMFPVLKVNVSGLDPNAMYSFLLDFVAADNHRWKYVNGEWVPGGKPEPQAPSCVYIHPDSPNFGAHWMKAPVSFSKVKLTNKLNGGGQIMLNSLHKYEPRIHIVRVGGPQRMITSHCFPETQFIAVTAYQNEEITALKIKYNPFAKAFLDAKERSDHKEMMEEPGDSQQPGYSQWGWLLPGTSTVCPPANPHPQFGGALSLPSTHSCDRYPTLRSHRSSPYPSPYAHRNNSPTYSDNSPACLSMLQSHDNWSSLGMPAHPSMLPVSHNASPPTSSSQYPSLWSVSNGAVTPGSQAAAVSNGLGAQFFRGSPAHYTPLTHPVSAPSSSGSPLYEGAAAATDIVDSQYDAAAQGRLIASWTPVSPPSMCoding sequence encoding Brachyury protein Isoform 1 with L254V.(1308 nt) SEQ ID NO: 8atgagctcccctggcaccgagagcgcgggaaagagcctgcagtaccgagtggaccacctgctgagcgccgtggagaatgagctgcaggcgggcagcgagaagggcgaccccacagagcgcgaactgcgcgtgggcctggaggagagcgagctgtggctgcgcttcaaggagctcaccaatgagatgatcgtgaccaagaacggcaggaggatgtttccggtgctgaaggtgaacgtgtctggcctggaccccaacgccatgtactccttcctgctggacttcgtggcggcggacaaccaccgctggaagtacgtgaacggggaatgggtgccggggggcaagccggagccgcaggcgcccagctgcgtctacatccaccccgactcgcccaacttcggggcccactggatgaaggctcccgtctccttcagcaaagtcaagctcaccaacaagctcaacggagggggccagatcatgctgaactccttgcataagtatgagcctcgaatccacatagtgagagttgggggtccacagcgcatgatcaccagccactgcttccctgagacccagttcatagcggtgactgcttatcagaacgaggagatcacagctcttaaaattaagtacaatccatttgcaaaggctttccttgatgcaaaggaaagaagtgatcacaaagagatgatggaggaacccggagacagccagcaacctgggtactcccaatgggggtggcncttcctggaaccagcaccgtttgtccacctgcaaatcctcatcctcagtttggaggtgccctctccctcccctccacgcacagctgtgacaggtacccaaccctgaggagccaccggtcctcaccctaccccagcccctatgctcatcggaacaattctccaacctattctgacaactcacctgcatgtttatccatgctgcaatcccatgacaattggtccagccttggaatgcctgcccatcccagcatgctccccgtgagccacaatgccagcccacctaccagctccagtcagtaccccagcctgtggtctgtgagcaacggcgccgtcaccccgggctcccaggcagcagccgtgtccaacgggctgggggcccagttcttccggggctcccccgcgcactacacacccctcacccatccggtctcggcgccctcttcctcgggatccccactgtacgaaggggcggccgcggccacagacatcgtggacagccagtacgacgccgcagcccaaggccgcctcatagcctcatggacacctgtgtcgccaccttccatgtga Brachyury-I3 fusion protein (449 aa)SEQ ID NO: 9 MKNNLYEEKMNMSKKSSPGTESAGKSLQYRVDHLLSAVENELQAGSEKGDPTERELRVGLEESELWLRFKELTNEMIVTKNGRRMFPVLKVNVSGLDPNAMYSFLLDFVAADNHRWKYVNGEWVPGGKPEPQAPSCVYIHPDSPNFGAHWMKAPVSFSKVKLTNKLNGGGQIMLNSLHKYEPRIHIVRVGGPQRMITSHCFPETQFIAVTAYQNEEITALKIKYNPFAKAFLDAKERSDHKEMMEEPGDSQQPGYSQWGWLLPGTSTVCPPANPHPQFGGALSLPSTHSCDRYPTLRSHRSSPYPSPYAHRNNSPTYSDNSPACLSMLQSHDNWSSLGMPAHPSMLPVSHNASPPTSSSQYPSLWSVSNGAVTPGSQAAAVSNGLGAQFFRGSPAHYTPLTHPVSAPSSSGSPLYEGAAAATDIVDSQYDAAAQGRLIASW TPVSPPSMCoding sequence encoding 13 Brachyury fusion protein of SEQ ID NO 9(1350 nt) SEQ ID NO: 10atgaaaaataacttgtatgaagamanatgaacatgagtaagaaaagctcccctggcaccgagagcgcgggaaagagcctgcagtaccgagtggaccacctgctgagcgccgtggagaatgagctgcaggcgggcagcgagaagggcgaccccacagagcgcgaactgcgcgtgggcctggaggagagcgagctgtggctgcgcttcaaggagctcaccaatgagatgatcgtgaccaagaacggcaggaggatgthccggtgctgaaggtgaacgtgtctggcctggaccccaacgccatgtactccttcctgctggacttcgtggcggcggacaaccaccgctggaagtacgtgaacggggaatgggtgccggggggcaagccggagccgcaggcgcccagctgcgtctacatccaccccgactcgcccaacttcggggcccactggatgaaggctcccgtctccttcagcaaagtcaagctcaccaacaagctcaacggagggggccagatcatgctgaactccttgcataagtatgagcctcgaatccacatagtgagagttgggggtccacagcgcatgatcaccagccactgcttccctgagacccagttcatagcggtgactgcttatcagaacgaggagatcacagctcttaaaattaagtacaatccatttgcaaaggctttccttgatgcaaaggaaagaagtgatcacaaagagatgatggaggaacccggagacagccagcaacctgggtactcccaatgggggtggcttcttcctggaaccagcaccgtttgtccacctgcaaatcctcatcctcagtttggaggtgccctctccctcccctccacgcacagctgtgacaggtacccaaccctgaggagccaccggtcctcaccctaccccagcccctatgctcatcggaacaattctccaacctattctgacaactcacctgcatgtttatccatgctgcaatcccatgacaattggtccagccttggaatgcctgcccatcccagcatgctccccgtgagccacaatgccagcccacctaccagctccagtcagtaccccagcctgtggtctgtgagcaacggcgccgtcaccccgggctcccaggcagcagccgtgtccaacgggctgggggcccagttcttccggggctcccccgcgcactacacacccctcacccatccggtctcggcgccctcttcctcgggatccccactgtacgaaggggcggccgcggccacagacatcgtggacagccagtacgacgccgcagcccaaggccgcctcatagcctcatggacacctgtgtcgccaccttccatgtgahCD4OL from NCBI RefSeq NP_000065.1. (261 amino acids) SEQ ID NO: 11MIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKENSFEMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLhCD4OL from NCBI RefSeq NP_000065.1. (789 nucleotides) SEQ ID NO: 12atgatcgagacatacaaccagacaagccctagaagcgccgccacaggactgcctatcagcatgaagatcttcatgtacctgctgaccgtgttcctgatcacccagatgatcggcagcgccctgtttgccgtgtacctgcacagacggctggacaagatcgaggacgagagaaacctgcacgaggacttcgtgttcatgaagaccatccagcggtgcaacaccggcgagagaagtctgagcctgctgaactgcgaggaaatcaagagccagttcgagggcttcgtgaaggacatcatgctgaacaaagaggaaacgaagaaagagaactccttcgagatgcagaagggcgaccagaatcctcagatcgccgctcacgtgatcagcgaggccagcagcaagacaacaagcgtgctgcagtgggccgagaagggctactacaccatgagcaacaacctggtcaccctggagaacggcaagcagctgacagtgaagcggcagggcctgtactacatctacgcccaagtgaccttctgcagcaacagagaggccagctctcaggctcctttcatcgccagcctgtgcctgaagtctcctggcagattcgagcggattctgctgagagccgccaacacacacagcagcgccaaaccttgtggccagcagtctattcacctcggcggagtgtttgagctgcagcctggcgcaagcgtgttcgtgaatgtgacagaccctagccaggtgtcccacggcaccggctttacatctttcggactgctgaagctgtgatgaSynthetic Her2 v2 amino acid sequence (1,145 amino acids): SEQ ID NO: 13MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELACPALVTYNTRTAKSMPNPEGRYTFGASCVTACPYNYLSTDAGACTLVCPAANQEVTAEDGTQRCEACSKACARVCYGLGMEHLREVRAVTSANIQEFAGCKKIEGSLAFLPESEDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPAADQCVACAHYKDPPACVARCPSGVKPDLSYMPIWAFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTSIISAVVGILLVVVLGVVFGILIKRRQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIMVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLGPASPLDSTFYRSLLEDDDMGDLVDAEEALVPQQGFFCPDPAPGAGGMVHHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMGAAKGLQSLPTHDPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPELGLDVPVSynthetic Her2 v2 nucleotide sequence (3441 nucleotides): SEQ ID NO: 14atggaactggctgctctgtgtagatggggactgctgcttgctctgttgcctcctggagctgcttctacccaagtgtgcacaggcaccgacatgaagctgagactgcctgcttctcctgagacacacctggacatgctgagacacctgtaccagggatgtcaggtggtgcagggaaatctggaactgacctacctgcctaccaacgccagcctgagctttctgcaggacatccaagaggtgcagggatacgtgctgatcgctcacaatcaagtgagacaggtgccactgcagaggctgagaatcgttagaggcacccagctgttcgaggacaactatgctctggctgtgctggacaatggcgaccctctgaacaacaccacacctgtgacaggagcttctcctggtggactgagagaactgcagctgagaagcctgaccgagatcctgaaaggaggagtgctgatccagcggaaccctcagctgtgctaccaggacaccatcctgtggaaggacatcttccacaagaacaaccagctggctctgacactgatcgacaccaacagaagcagagcctgccatccttgctctcccatgtgcaagggctctagatgttggggagagagcagcgaggattgccagagcctgaccagaacagtgtgtgctggaggatgtgccagatgcaaaggacctctgcctaccgactgctgccacgagcaatgtgcagctggatgtacaggaccaaagcactctgattgcctggcctgcctgcacttcaaccactctggaatctgcgagctcgcctgtcctgctctggtcacctacaacacacggaccgccaagagcatgcctaatcctgaaggcagatacacctttggagccagctgtgtgacagcctgtccttacaactacctgagcaccgacgctggagcctgcacactcgtttgtcctgctgccaatcaagaagtgacggccgaggacggcacccagagatgcgaggcctgtagcaaggcttgcgctagagtgtgttacggactcggcatggaacacctgagagaagtgagagccgtgaccagtgccaacatccaagagtttgctggctgcaagaagatctttggcagcctcgccttcctgcctgagagcttcgatggcgatcctgccagcaatactgctcctctgcagcctgaacagctccaggtgttcgagacactggaagagatcacaggctacctgtacatcagcgcatggccagacagcctgcctgacctgtccgtgttccagaacctgcaagtgatcagaggcagaatcctgcacaacggagcctattctctgaccctgcaaggcctgggaatcagctggctgggactgagatccctgagagagcttggatctggcctggctctgatccaccacaatacccacctgtgcttcgtgcacaccgtgccttgggaccagctgtttcggaatcctcatcaggctctgctgcacacagccaacagacctgaggatgagtgtgttggcgaaggcctggcttgtcaccagctctgtgctagaggacactgttggggacctggacctacacagtgtgtgaactgtagccagttcctgagaggccaagaatgcgtggaagagtgtagagttctgcagggactgcctcgcgagtacgtgaacgctagacactgtctgccttgtcatcccgagtgccagcctcagaatggcagcgtgacatgttttggaccagctgccgatcagtgcgtggcctgtgctcactataaggaccctccagcctgcgtggccagatgtcctagcggagtgaagcctgacctgagctacatgcccatctgggcatttccagatgaggaaggagcttgccagccttgtcctatcaactgcacccacagctgcgtggacctggacgataagggatgtccagccgagcagagagcctctccactgacctctatcatctctgccgtcgtgggcatcctgctggtggtggttctgggagttgtgttcggcatcctgatcaagagacggcagcagaagatccggaagtacaccatgcggagactgctgcaagagactgagctggtggaacctctgacacccagcggagctatgcctaaccaggctcagatgcggattctgaaagaaaccgagctgcggaaagtgaaggtgctcggctctggagcctttggcacagtgtacaaaggcatctggatccctgacggagagaacgtgaagattcctgtggccatcatggtgctgagagagaacacaagtcccaaggccaacaaagagatcctggacgaggcctacgtgatggctggtgttggcagcccttatgtgtctagactgctgggcatctgtctgaccagcaccgtgcagctggtcactcagctgatgccttacggctgcctgctggatcacgtgagagagaatagaggcagactgggctctcaggacctgctgaactggtgcatgcagatcgccaagggcatgagctacctcgaggatgtgagactggtccacagagatctggctgccagaaacgtgctcgtgaagtctcctaaccacgtgaagatcaccgacttcggactggctaggctgctggatatcgacgagacagagtaccacgctgatggaggcaaggtgcccatcaagtggatggctctggaatccatcctgagacggagattcacccaccagtccgatgtgtggtcttacggagtgacagtgtgggagctgatgaccttcggagccaagccttacgacggcatccctgccagagagatcccagatctgctggaaaagggagagagactgcctcagcctcctatctgcaccatcgacgtgtacatgattatggtcaagtgttggatgatcgacagcgagtgcagacccagattcagagaactggtgtccgagttctctcggatggccagagatcctcagagattcgtggtcatccagaacgaggatctgggacctgccagccctctggacagcaccttctacagatccctgctggaagatgacgacatgggtgacctggtggacgctgaagaagctctggttcctcagcagggcttcttctgccctgatcctgctccaggagcaggtggaatggtgcatcacagacacagaagctccagcaccagaagcggaggcggagatctgacactgggactcgagccatctgaggaagaggctcctagatctcctctggctccttctgaaggagctggaagcgacgttttcgacggagatcttggaatgggagctgccaaaggactccagtctctgcccacacacgacccatctccactgcagagatacagcgaggaccctaccgtgcctctgccaagcgagacagatggatatgtggcacctctgacctgctctcctcagccagaactgggacttgatgtgcctgtttgatgaSynthetic Twist amino acid sequence (205 amino acids): SEQ ID NO: 15MQDVSSSPVSPADDSLSNSEEEPDRQQPASGKRGARKRRSSRRSAGGSAGPGGATGGGIGGGDEPGSPAQGKRGKKSAGGGGGGGAGGGGGGGGGSSSGGGSPQSYEELQTQRVMANVRERQRTQSLNEAFAALRKIIPTLPSDKLSKIQTLKLAARYIDFLYQVLQSDELDSKMASCSYVAHERLSYAFSVWRMEGAWSMSASHSynthetic Twist nucleotide sequence (618 nucleotides): SEQ ID NO: 16atgcaggacgtgtccagcagccctgtgtctcctgccgacgacagcctgagcaacagcgaggaagaacccgacagacagcagcccgcctctggcaagagaggcgccagaaagagaagaagctccagaagaagcgctggcggctctgctggacctggcggagctacaggcggaggaattggaggcggagatgagcctggctctccagcccagggcaagaggggcaagaaatctgctggcggaggcggcggaggaggagctggaggcggaggaggaggcggcggaggatcaagttctggcggaggaagccctcagagctacgaggaactgcagacccagcgcgtgatggccaacgtgcgcgagagacagagaacccagagcctgaacgaggccttcgccgccctgagaaagatcatccccaccctgcccagcgacaagctgagcaagatccagaccctgaagctggccgccagatatatcgacttcctgtatcaagtgctgcagagcgacgagctggacagcaagatggccagctgctcctacgtggcccacgagagactgagctacgccttcagcgtgtggcggatggaaggcgcctggtctatgagcgccagccactgaSynthetic murine CD4OL amino acid sequence (260 amino acids):SEQ ID NO: 17MIETYSQPSPRSVATGLPASMKIFMYLLTVFLITQMIGSVLFAVYLHRRLDKVEEEVNLHEDFVFIKKLKRCNKGEGSLSLLNCEEMRRQFEDLVKDITLNKEEKKENSFEMQRGDEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVMLENGKQLTVKREGLYYVYTQVTFCSNREPSSQRPFIVGLWLKPSSGSERILLKAANTHSSSQLCEQQSVHLGGVFELQAGASVFVNVTEASQVIHRVGFSSFGLLKLSynthetic murine CD4OL nucleotide sequence (786 nucleotides):SEQ ID NO:18atgatcgagacatacagccagcccagccccagaagcgtggccacaggactgcctgccagcatgaagatctttatgtacctgctgaccgtgttcctgatcacccagatgatcggcagcgtgctgttcgccgtgtacctgcacagacggctggacaaggtggaagaggaagtgaacctgcacgaggacttcgtgttcatcaagaaactgaagcggtgcaacaagggcgagggcagcctgagcctgctgaactgcgaggaaatgagaaggcagttcgaggacctcgtgaaggacatcaccctgaacaaagaggaaaagaaagaaaactccttcgagatgcagaggggcgacgaggaccctcagatcgctgctcacgtggtgtccgaggccaacagcaacgccgcttctgtgctgcagtgggccaagaaaggctactacaccatgaagtccaacctcgtgatgctggaaaacggcaagcagctgacagtgaagcgcgagggcctgtactatgtgtacacccaagtgacattctgcagcaacagagagcccagcagccagaggcccttcatcgtgggactgtggctgaagcctagcagcggcagcgagagaatcctgctgaaggccgccaacacccacagcagctctcagctgtgcgagcagcagagcgtgcacctgggcggagtgttcgagctgcaagctggcgcctccgtgttcgtgaacgtgacagaggccagccaagtgatccacagagtgggcttcagcagctttggactgctgaaactgtaatga

REFERENCES

The references included as part of the present disclosure, in additionto those listed below, are hereby incorporated by reference in theirentirety: World Health Organization, World Health report (2013); Torre,“Global Cancer Statistics” (2012) CA: A Cancer Journalfor Clinicians;Ross (2003), “The Her-2/neu gene and protein in breast cancer 2003:biomarker and target of therapy,” Oncologist; Palena (2007) Clin. CancerRes., “The human T-box mesodermal transcription factor Brachyury is acandidate target for T-cell-mediated cancer immunotherapy”; Hynes andLane (2005) Nat. Rev. Cancer, “ERBB receptors and cancer: the complexityof targeted inhibitors”; Cho (2003) Nature, “Structure of theextracellular region of HER2 alone and in complex with the HerceptinFab”; Satyanarayanajois (2009) Chem. Biol. Drug Des., “Design,Synthesis, and Docking Studies of Peptidomimetics based onHER2-Herceptin Binding Site with Potential Antiproliferative ActivityAgainst Breast Cancer Cell lines”; Franklin (2004) Cancer Cell,“Insights into ErbB signaling from the structure of the ErbB2-pertuzumabcomplex”; Yang (2009), Paper 391, “Targeting The Dimerization Of ERBBReceptor,” All Theses and Dissertations; Tan (2005) Cancer Res., “ErbB2promotes Src synthesis and stability: novel mechanisms of Src activationthat confer breast cancer metastasis”; Roskoski (2014) Pharmacol. Res.“ErbB/HER protein-tyrosine kinases: Structures and small moleculeinhibitors”; Roselli (2012) Clin. Cancer Res., “Brachyury, a driver ofthe epithelial-mesenchymal transition, is overexpressed in human lungtumors: an opportunity for novel interventions against lung cancer”;Stoller and Epstein (2005) Hum. Mol. Genet., “Identification of a novelnuclear localization signal in Tbx1 that is deleted in DiGeorge syndromepatients harboring the 1223delC mutation”; Lauterbach (2013) Front.Immunol., “Genetic Adjuvantation of Recombinant MVA with CD40LPotentiates CD8 T Cell Mediated Immunity”; Guardino et al. (2009) CancerRes. 69 (24 Supp): Abstract nr 5089, “Results of Two Phase I ClinicalTrials of MVA-BN®-HER2 in HER-2 Overexpressing Metastatic Breast CancerPatients”; Heery et al. (2015) J. Immunother. Cancer 3 (Suppl. 2): P132,“Phase I, dose-escalation, clinical trial of MVA-Brachyury-TRICOMvaccine demonstrating safety and brachyury-specific T cell responses”;Brodowicz et al. (2001) Br. J. Cancer, “Anti-Her-2/neu antibody inducesapoptosis in Her-2/neu overexpressing breast cancer cells independentlyfrom p53 status”; Stackaruk et al. (2013) Expert Rev. Vaccines12(8):875-84, “Type I interferon regulation of natural killer cellfunction in primary and secondary infections”; Muller et al. (2017)Front. Immunol. 8: 304, “Type I Interferons and Natural Killer CellRegulation in Cancer”; Yamashita et al. (January 2016) ScientificReports 6 (Article number 19772), “A novel method for evaluatingantibody dependent cell-mediated cytotoxicity by flow cytometry usinghuman peripheral blood mononuclear cells”; Broussas et al. (2013)Methods Mol. Biol. 988: 305-17, “Evaluation of antibody-dependent cellcytotoxicity using lactate dehydrogenase (LDH) measurement”; Tay et al.(2016) Hum. Vaccines and Immunother. 12: 2790-96, “TriKEs and BiKEs joinCARs on the cancer immunotherapy highway”; and Kono et al. (2004) Clin.Cancer Res. 10: 2538-44, “Trastuzumab (Herceptin) Enhances ClassI-Restricted Antigen Presentation Recognized by Her2/neu Specific TCytotoxic Lymphocytes.”

We claim:
 1. A method of inducing both an innate and adaptive immuneresponse in a patient comprising (a) administering intravenously to saidpatient a recombinant modified vaccinia virus Ankara (MVA) comprising:(i) a first nucleic acid encoding a first heterologous tumor-associatedantigen (TAA); and (ii) a second nucleic acid encoding CD40L; and (b)administering to said patient an antibody that comprises an Fc domainand is specific to an antigen expressed on the cell membrane of a tumorcell; whereby an enhanced Natural Killer (NK) cell response and anenhanced T cell response is induced in said patient.
 2. The method ofclaim 1, wherein said first nucleic acid encodes a tumor-associatedantigen that is HER2.
 3. The method of claim 2, wherein the antibody ofstep (b) is trastuzumab and the HER2 antigen comprises at least onemutation in the trastuzumab binding domain and selected from the groupconsisting of: E580, D582, P594, F595, K615, and Q624 corresponding tothe amino acid sequence set forth in SEQ ID NO:13.
 4. The method ofclaim 2, wherein the antibody of step (b) is pertuzumab and the HER2antigen comprises at least one mutation in the pertuzumab binding domainand selected from the group consisting of: H267, Y274, F279, V308, S310,L317, H318, K333, and P337 corresponding to the amino acid sequence setforth in SEQ ID NO:13.
 5. The method of claim 2, wherein the HER2antigen comprises at least one mutation selected from the groupconsisting of: (a) a mutation that prevents extracellular dimerizationof HER2 that is D277R or E280K; (b) a mutation that prevents tyrosinekinase activity of HER2 that is K753M; and (c) a mutation thatinterferes with phosphorylation of HER2 that is Y1023A or is thedeletion of residues 1139-1248.
 6. The method of claim 2, wherein saidfirst nucleic acid encodes a HER2 antigen having the amino acid sequenceset forth in SEQ ID NO:1 or SEQ ID NO:
 13. 7. The method of claim 2,wherein said recombinant modified vaccinia virus Ankara (MVA) furthercomprises a third nucleic acid encoding a heterologous tumor-associatedantigen (TAA) which is Brachyury.
 8. The method of claim 7, wherein saidBrachyury antigen comprises one or more mutations to the nuclearlocalization signal (NLS) domain or in which the NLS domain is deleted.9. The method of claim 8, wherein said Brachyury antigen has an aminoacid sequence that comprises the sequence set forth in SEQ ID NO:3.